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BIOSYNTHESIS OF CYCLIC PEPTIDE NATURAL PRODUCTS
IN MUSHROOMS
By
Robert Michael Sgambelluri
A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Biochemistry & Molecular Biology – Doctor of Philosophy
2017
ABSTRACT
BIOSYNTHESIS OF CYCLIC PEPTIDE NATURAL PRODUCTS
IN MUSHROOMS
By
Robert Michael Sgambelluri
Cyclic peptide compounds possess properties that make them attractive candidates in the
development of new drugs and therapeutics. Mushrooms in the genera Amanita and Galerina
produce cyclic peptides using a biosynthetic pathway that is combinatorial by nature, and
involves an unidentified, core set of tailoring enzymes that synthesize cyclic peptides from
precursor peptides encoded in the genome. The products of this pathway are collectively referred
to as cycloamanides, and include amatoxins, phallotoxins, peptides with immunosuppressant
activities, and many other uncharacterized compounds. This work aims to describe cycloamanide
biosynthesis and its capacity for cyclic peptide production, and to harness the pathway as a
means to design and synthesize bioactive peptides and novel compounds.
The genomes of Amanita bisporigera and A. phalloides were sequenced and genes encoding
cycloamanides were identified. Based on the number of genes identified and their sequences, the
two species are shown to have a combined capacity to synthesize at least 51 unique
cycloamanides. Using these genomic data to predict the structures of uncharacterized
cycloamanides, two new cyclic peptides, CylE and CylF, were identified in A. phalloides by
mass spectrometry. Two species of Lepiota mushrooms, previously not known to produce
cycloamanides, were also analyzed and shown to contain amatoxins, the toxic cycloamanides
responsible for fatal mushroom poisonings. The mushroom Galerina marginata, which also
produces amatoxins, was used as a model orgasnism for studying cycloamanide biosynthesis due
to its culturability. Three enzymes involved in the biosynthesis of cycloamanides were identified
in gene knockout studies: a predicted flavin-containing monooxygenase (FMO), P450
monooxygenase, and prolyl oligopeptidase (POP). The gene encoding a specific predicted prolyl
oligopeptidase (POPB) was cloned and expressed in Saccharomyces cerevisiae for further
characterization, and in vitro studies revealed that the enzyme is bifunctional, catalyzing both a
hydrolysis reaction and the key cyclization step in cycloamanide biosynthesis. The utility of
POPB as a general catalyst for peptide cyclization was explored by defining its subtrate
preferences and limitations. POPB was shown to be highly versatile, catalyzing cyclization of
diverse peptide sequences ranging from 8-16 residues in length and sequences containing
modified amino acids in addition to the proteinogenic twenty. A method for the use of POPB for
the production of combinatorial cyclic peptide libraries is also presented. A total of 100 cyclic
peptides, including both novel compounds and bioactive cycloamanides, were produced in these
studies and demonstrate the applications of POPB in biotechnology.
Copyright by
ROBERT MICHAEL SGAMBELLURI
2017
v
TABLE OF CONTENTS
LIST OF TABLES ........................................................................................................... viii
LIST OF FIGURES ........................................................................................................... ix
KEY TO ABBREVIATIONS .......................................................................................... xiii
CHAPTER 1 INTRODUCTION ........................................................................................1
1.1 Cyclic Peptides ................................................................................................2
1.2 Cycloamanides ................................................................................................5
1.3 Ribosomal Biosynthesis of Cycloamanides ....................................................8
WORKS CITED ...................................................................................................11
CHAPTER 2 DETECTION AND PROFILING OF AMATOXINS IN LEPIOTA
MUSHROOMS ..................................................................................................................16
2.1 Abstract .........................................................................................................17
2.2 Introduction ...................................................................................................18
2.3 Methods .........................................................................................................21
2.3.1 Mushroom Collection and Identification .........................................21
2.3.2 Toxin Extraction and LCMS ............................................................22
2.4 Results ...........................................................................................................23
2.4.1 Toxins in Amanita and Galerina Mushrooms .................................23
2.4.2 Toxins in Lepiota Mushrooms .........................................................24
2.5 Discussion .....................................................................................................27
APPENDIX ..........................................................................................................28
WORKS CITED ...................................................................................................31
CHAPTER 3 GENOMIC CAPACITY FOR CYCLOAMANIDE BIOSYNTHESIS
IN AMANITA MUSHROOMS .........................................................................................34
3.1 Abstract .........................................................................................................35
3.2 Introduction ...................................................................................................36
3.3 Methods .........................................................................................................37
3.3.1 Genomics and Transcriptomics........................................................37
3.3.2 LC/MS/MS of Predicted Cycloamanides.........................................37
3.4 Results ...........................................................................................................39
3.4.1 MSDIN Genes in Amanita bisporigera and A. phalloides ..............39
3.4.2 New Cycloamanides in Amanita phalloides ....................................39
3.5 Discussion .....................................................................................................43
APPENDIX ..........................................................................................................45
WORKS CITED ...................................................................................................49
vi
CHAPTER 4 CHARACTERIZATION OF AMANITIN BIOSYNTHESIS IN
GALERINA MARGINATA ..............................................................................................52
4.1 Abstract .........................................................................................................53
4.2 Introduction ...................................................................................................54
4.3 Methods .........................................................................................................57
4.3.1 Galerina Growth and Toxin Analysis ..............................................57
4.3.2 Galerina Transformation and Gene Knockouts ...............................58
4.3.3 Purification of an Amanitin Intermediate and NMR .......................58
4.3.4 Analysis of Gene Expression by RT-PCR .......................................59
4.4 Results ...........................................................................................................60
4.4.1 Time Course of Amanitin Production ..............................................60
4.4.2 Genes Involved in Amanitin Biosynthesis .......................................61
4.4.3 Role of a P450 Monooxygenase in Amanitin Biosynthesis.............64
4.4.4 Regulation of Biosynthetic Genes ...................................................66
4.5 Discussion .....................................................................................................68
APPENDIX ..........................................................................................................70
WORKS CITED ...................................................................................................78
CHAPTER 5 BIOCHEMICAL CHARACTERIZATION OF PROLYL
OLIGOPEPTIDASE B AS A PEPTIDE MACROCYCLASE .........................................81
5.1 Abstract .........................................................................................................82
5.2 Introduction ...................................................................................................83
5.3 Methods .........................................................................................................85
5.3.1 Protein Expression and Purification.................................................85
5.3.2 Enzyme Assays ................................................................................86
5.3.3 Product Purification and NMR Spectroscopy ..................................86
5.4 Results ...........................................................................................................88
5.4.1 Preparation of Recombinant GmPOPB ...........................................88
5.4.2 GmPOPB Catalyzes Peptide Macrocyclization ...............................89
5.4.3 GmPOPB is a Bifunctional Enzyme ................................................91
5.4.4 Residues Involved in Macrocyclization ...........................................94
5.5 Discussion .....................................................................................................96
APPENDIX ..........................................................................................................97
WORKS CITED .................................................................................................104
CHAPTER 6 VERSATILITY OF PROLYL OLIGOPEPTIDASE B IN PEPTIDE
MACROCYCLIZATION ................................................................................................108
6.1 Abstract .......................................................................................................109
6.2 Introduction .................................................................................................110
6.3 Methods .......................................................................................................112
6.3.1 DNA Constructs .............................................................................112
6.3.2 Preparation of POPB Substrates ....................................................112
6.3.3 Cyclization Assays and LCMS ......................................................113
6.3.4 Library Preparation and Analysis ..................................................113
6.4 Results .........................................................................................................115
6.4.1 Enzyme and Substrate Preparation ................................................115
vii
6.4.2 Amino Acid Preferences for Cyclization .......................................115
6.4.3 Cyclization of Sequences Containing Unusual Amino Acids .......118
6.4.4 Core Domain Length Requirement ................................................120
6.4.5 Synthesis of Naturally Occurring Cycloamanides .........................120
6.4.6 Cyclization of the Phalloidin Sequence with D-threonine .............123
6.4.7 Cyclic Peptide Library Production.................................................123
6.5 Discussion ...................................................................................................127
APPENDIX ........................................................................................................128
WORKS CITED .................................................................................................145
viii
LIST OF TABLES
Table 2.1: Compounds Identified in Extracts of Amanita, Galerina and Lepiota
Mushrooms .......................................................................................................................24
Table 2.2: α-Amanitin Concentrations in Mushrooms .................................................26
Table 3.1: Cycloamanide Sequences in the Genomes of A. bisporigera and
A. phalloides ......................................................................................................................40
Table S3.1: List of Core Domains Identified Among MSDIN Transcripts from
RNAseq of Amanita bisporigera ......................................................................................46
Table S3.2: Alphabetical List of All Unique MSDIN Core Sequences Identified
to Date ...............................................................................................................................47
Table S4.1: Table of all 2D NMR Correlations Observed in the Amanitin
Intermediate .....................................................................................................................76
Table 5.1: GmPOPB Cyclization Kinetic Constants with 35mer and 25mer
GmAMA1 Substrates.......................................................................................................93
Table 5.2: Differentially Conserved Residues between POPA and POPB .................95
Table 6.1: Tolerance of POPB for Amino Acid Substitutions in the Core Region
of AMA1 ..........................................................................................................................117
Table 6.2: Cyclization of Naturally Occurring Cycloamanides.................................122
Table S6.1: Compared Cyclization Yields of 35mer and 25mer Substrates ............129
Table S6.2: Alphabetical List of Cyclic Peptides Produced with POPB ...................143
Table S6.3: Alphabetical List of Peptides Not Efficiently Cyclized by POPB .........144
ix
LIST OF FIGURES
Figure 1.1: Macrocyclic Bonds in Cyclic Peptides. .........................................................2
Figure 1.2: RiPP Precursor Peptide Structure................................................................4
Figure 1.3: Amatoxin and Phallotoxin Structure............................................................6
Figure 1.4: Other Cycloamanide Compounds.................................................................7
Figure 1.5: Southern Blot Analysis of Amanita Mushrooms. ........................................9
Figure 1.6: Primary Structure of the Cycloamanide Precursor Peptides. ...................9
Figure 1.7: Compared Sequences of Amanitin Precursors from Amanita
and Galerina .....................................................................................................................10
Figure 2.1: Amatoxin and Phallotoxin Families of Bicyclic Peptides .........................19
Figure 2.2: Mushroom Species Analyzed in this Study for Toxin Content ................20
Figure 2.3: Toxin Profiles of Amanita, Galerina, and Lepiota Mushrooms ................25
Figure S2.1: ITS Sequences from Species of Lepiota Mushrooms...............................29
Figure 3.1: MS/MS Analysis of Cycloamanide E ..........................................................41
Figure 3.2: MS/MS Analysis of Cycloamanide F ..........................................................42
Figure 3.3: Amino Acid Frequencies in MSDIN Core Domain Sequences ................44
Figure 4.1: Basidiocarps of Galerina marginata ............................................................54
Figure 4.2: Steps in the Biosynthesis of α-Amanitin from the GmAMA1
Precursor Peptide.............................................................................................................56
Figure 4.3: Genes Adjacent to GmAMA1 in the Galerina marginata Genome
with Relevant Predicted Functions.................................................................................56
Figure 4.4: Culture of Galerina marginata Mycelium. .................................................60
Figure 4.5: Time Course of α-Amanitin Production in Galerina marginata
Cultures. ............................................................................................................................61
x
Figure 4.6: POPB Knockout in Galerina and Effects on α-Amanitin Production. ....62
Figure 4.7: FMO Knockout in Galerina and Effects on α-Amanitin Production.......63
Figure 4.8: P450 Knockout in Galerina and Effects on α-Amanitin Production. ......63
Figure 4.9: Compared Structures of α-Amanitin and the Pathway Intermediate
Purified from the P450(-) Strain of G. marginata . .......................................................64
Figure 4.10: Compared 1H-
13C HSQC Spectra of α-Amanitin and the
Intermediate . ...................................................................................................................65
Figure 4.11: Expression of Genes Involved in Amanitin Biosynthesis. .......................67
Figure S4.1: 1H Spectra of α-Amanitin and the Pathway Intermediate Purified
from a P450(-) Strain of G. marginata ............................................................................71
Figure S4.2: 2D COSY Spectrum of the Amanitin Intermediate ................................72
Figure S4.3: 2D TOCSY Spectrum of the Amanitin Intermediate .............................72
Figure S4.4: 2D ROESY Spectrum of the Amanitin Intermediate .............................73
Figure S4.5: 1H-
13C HSQC Spectrum of the Amanitin Intermediate .........................73
Figure S4.6: 1H-
13C HMBC Spectrum of the Amanitin Intermediate ........................74
Figure S4.7: Key HMBC Correlations Observed in the Amanitin Intermediate
for Structure Determination ...........................................................................................75
Figure 5.1: Crystal Structure of Prolyl Oligopeptidase from Porcine Brain. ............84
Figure 5.2: Purification of Recombinant GmPOPB Expressed in Yeast ....................88
Figure 5.3: Time Course of Conversion of GmAMA1 to cyclo-IWGIGCNP .............89
Figure 5.4: Amide Bond Couplings in HMBC Spectrum of cyclo-IWGIGCNP ........90
Figure 5.5: Reaction Products from GmPOPB Activity on GmAMA1 ......................92
Figure 5.6: Two-Step Nonprocessive Reaction Catalyzed by POPB on the
α-Amanitin Precursor Peptide ........................................................................................92
Figure 5.7: Kinetic Analysis of GmPOPB ......................................................................93
Figure 5.8: Hyopthetical Mechanism for Macrocyclization Catalyzed by POPB .....96
xi
Figure S5.1: 2D gCOSY Spectrum of cyclo-IWGIGCNP ............................................98
Figure S5.2: 2D TOCSY Spectrum of cyclo-IWGIGCNP ...........................................98
Figure S5.3: Amide Region from TOCSY Spectrum of cyclo-IWGIGCNP ...............99
Figure S5.4: 2D ROESY Spectrum of cyclo-IWGIGCNP .........................................100
Figure S5.5: 2D 1H-
13C HSQC Spectrum of cyclo-IWGIGCNP................................100
Figure S5.6: Formation of cyclo-IWGIGCNP Product from 35mer and 25mer
Substrates........................................................................................................................101
Figure S5.7: Multiple Sequence Alignment of POPB and Other Prolyl
Oligopeptidases ..............................................................................................................102
Figure 6.1: Expression and Purification of the Amanitin Precursor Peptide ..........116
Figure 6.2: Cyclization of Peptides Containing Unusual Amino Acids ....................119
Figure 6.3: POPB Products Produced from Substrates with Varying Core Domain
Lengths ............................................................................................................................121
Figure 6.4: LCMS Comparing POPB Products Produced from Substrates
Containing the PHD Sequence with Either L-Thr or D-Thr .....................................124
Figure 6.5: Scheme for Generating Mixed Cyclic Peptide Libraries ........................125
Figure S6.1: Effect of Single Amino Acid Substitutions on Cyclization by POPB
at Position 1 of the AMA1 Core Domain .....................................................................129
Figure S6.2: Effect of Single Amino Acid Substitutions on Cyclization by POPB
at Position 2 of the AMA1 Core Domain .....................................................................130
Figure S6.3: Effect of Single Amino Acid Substitutions on Cyclization by POPB
at Position 3 of the AMA1 Core Domain .....................................................................131
Figure S6.4: Effect of Single Amino Acid Substitutions on Cyclization by POPB
at Position 4 of the AMA1 Core Domain .....................................................................132
Figure S6.5: Effect of Single Amino Acid Substitutions on Cyclization by POPB
at Position 5 of the AMA1 Core Domain .....................................................................133
Figure S6.6: Effect of Single Amino Acid Substitutions on Cyclization by POPB
at Position 6 of the AMA1 Core Domain .....................................................................134
xii
Figure S6.7: Effect of Single Amino Acid Substitutions on Cyclization by POPB
at Position 7 of the AMA1 Core Domain .....................................................................135
Figure S6.8: LCMS Traces of Naturally Occurring Cycloamanide Core Regions
Cyclized by POPB ..........................................................................................................136
Figure S6.9: Simultaneous Production of Ten Cyclic Peptides Using POPB ...........137
Figure S6.10: Batch Production of Cyclic Peptides Using POPB ..............................138
xiii
KEY TO ABBREVIATIONS
ACN Acetonitrile, CH3CN
BCA Bicinchoninic acid
BLAST Basic local alignment search tool
BSA Bovine serum albumin
CTAB Cetyltrimethyl ammonium bromide
DQF-COSY Double quantum filtered correlation spectroscopy
DMSO Dimethyl sulfoxide
DTT Dithiothreitol
EIC Extracted ion chromatogram
ESI Electrospray ionization
FMO Flavin-containing monooxygenase
HSQC Heteronuclear single-quantum correlation spectroscopy
HMBC Heteronuclear multiple-bond correlation spectroscopy
HPLC High-performance liquid chromatography
IPTG Isopropyl β-D-1-thiogalactopyranoside
ITS Internal transcribed spacer
LCMS Liquid chromatography - mass spectrometry
LC/MS/MS Liquid chromatography - tandem mass spectrometry
MBP Maltose-binding protein
MS Mass spectrometry
NOE Nuclear Overhauser effect
xiv
NRPS Nonribosomal peptide synthetase
OATP Organic anion-transporting polypeptide
PCR Polymerase chain reaction
PDA Potato Dextrose Agar
PDB Protein Data Bank
POP Prolyl oligopeptidase
PTM Post-translational modification
RiPP Ribosomally synthesized and post-translationally modified peptide
ROESY Rotating frame nuclear Overhauser effect spectroscopy
RT-PCR Reverse transcription polymerase chain reaction
SDS-PAGE Sodium dodecyl sulfate - polyacrylamide gel electrophoresis
tblastn BLAST query of protein sequence against translated nucleotide
TBS Tris-buffered saline
TLC Thin layer chromatography
TOCSY Total correlation spectroscopy
1
CHAPTER 1
INTRODUCTION
2
1.1 Cyclic Peptides
Cyclic peptides are compounds composed of amino acids with covalent linkages forming
macrocyclic ring structures [1]. Familiar examples include the immunosuppressant cyclosporine
from fungi [2], the peptide hormone oxytocin [3,4], and the antibiotic daptomycin from
Streptomyces roseosporus [5]. Macrocycles may be formed by linkages between the amino and
carboxyl termini, between amino acid side-chains, or by a linkage between an N/C-terminus and
side-chain (Figure 1.1). Cyclic peptides are produced by prokaryotes and eukaryotes and are
abundant and diverse in nature, ranging in size from the 6-11 residue cyanobactins found in
cyanobacteria [6] to the 35-78 residue bacteriocins found in bacteria [7].
Figure 1.1: Macrocylic Bonds in Cyclic Peptides. The characteristic ring structures of
cyclic peptides arise from covalent bonds linking amino acid side-chains, the N- and C-
termini, or a side-chain to an N/C-terminus.
Macrocyclic bonds afford peptides with three attributes than result in a high potential for
bioactivity. First, cyclic peptides bind to targets with high affinity that results from their
increased structural rigidity and a reduced entropic penalty to binding [8]. Second, cyclic
3
peptides are generally stable against proteolysis since macrocyclic bonds involving the N- or C-
termini prevent exoprotease activity, and because any macrocyclic bond is conformationally
restrictive. Fairlie et al (2000) [9] compared the conformations of 266 bound protease substrates
and inhibitors from structures in the Protein Data Bank (PDB) and found that all adopted the
same extended conformation. The structural constraints imposed by cyclization in peptides often
prevents this extended conformation that is required for protease activity [1,9]. Finally, enhanced
membrane permeability is often displayed by cyclic peptides, and is proposed to result from their
ability to more readily adopt conformations that bury polar backbone and side-chain groups in
intramolecular interactions [10]. This is exemplified in cyclosporine A, which contains backbone
amides and carbonyls that form intramolecular hydrogen bonds in nonpolar solvent [11] but that
are solvent-exposed in water [12], affording cell permeability and broad solubility in different
solvents [13].
These attributes that often result in bioactivity make cyclic peptides desirable candidates in the
development of new therapeutics and research tools [14]. Since 2006, nine cyclic peptides have
been approved for clinical use and at least 20 are currently being evaluated for the treatment of
infections, cancer, metabolic disorders, blood disorders, and cardiovascular disease [15].
Currently, more than 40 cyclic peptides are in clinical use and most are derived from natural
products [13,15]. Despite their usefulness, however, organic synthesis of these compounds
remains difficult and expensive [16]. Nature has overcome these challenges through the use of
nonribosomal peptide synthetase (NRPS) enzymes [17], as well as recently discovered ribosomal
pathways, in which genetically encoded precursor peptides (Figure 1.2) serve as substrates for
promiscuous tailoring enzymes resulting in natural libraries of cyclic peptides [18]. Peptide
natural products arising from ribosomal pathways are referred to as ribosomally synthesized and
4
post-translationally modified peptides, or RiPPs [19]. Macrocyclic RiPP natural products are
now known to be widespread throughout nature, and examples include the microcins [20] and
thiopeptides [21] in bacteria, cyanobactins in cyanobacteria [6], conotoxins in conesnails [22],
cyclotides in plants [23], and ustiloxins [24] and cycloamanides [25] in fungi. While an extensive
number of ribosomal cyclic peptides with useful bioactivities have been discovered, many of the
enzymes involved in their biosynthesis remain uncharacterized and elucidation of these pathways
could provide efficient strategies for producing these compounds in sufficient quantities. In
addition, since RiPP tailoring enzymes display broad substrate preferences and the amino acid
sequences of the products can be easily manipulated at the DNA level, RiPP pathways are well
suited for use in synthetic biology and the production of novel compounds.
Figure 1.2: RiPP Precursor Peptide Structure. Ribosomal peptide precursors are
generally composed of leader, core, and recognition or follower domains. The core
peptide contains the amino acids found in the final RiPP product.
5
1.2 Cycloamanides
Cycloamanides are cyclic peptides produced by mushrooms in the genus Amanita and include
amatoxins, the causative agents of fatal mushroom poisonings [26]. Amatoxins bind to and
inhibit eukaryotic RNA polymerase II [27] and are often employed as experimental tools for the
study of their target. α-Amanitin, the most abundant and potent of the amatoxins, displays an oral
LD50 of 0.1 mg/kg in rats [26] and the crystal structure of RNA polymerase II with bound
amanitin has been solved [28]. Stability of the amatoxins against cooking and the digestive tract,
as well as rapid uptake into hepatocytes through the OATP transporter protein [29] contribute to
the toxicity of these compounds. Phallotoxins comprise another family of cycloamanides, and
include phalloidin, which binds with high affinity to actin [30]. Fluorescent conjugates of
phalloidin are used in cell imaging [31], and the compound is one of the most widely used tools
in chemical biology.
Structurally, the amatoxins are bicyclic octapeptides with the amino acid sequence
IWGIGC(N/D)P, and the phallotoxins are bicyclic heptapeptides with the sequence
AWL(V/A)(D/T)CP (Figure 1.3). Both the amatoxins and phallotoxins contain cyclic
backbones, with N- and C-termini linked ‘head-to-tail’ by an additional peptide bond [26]. Other
modifications found in both toxin families include side-chain hydroxylations and a unique
“tryptathionine” [32] linkage between the side-chains of tryptophan and cysteine, a modification
that has not been seen in other natural products, resulting in the overall bicyclic structure.
Phallotoxins also contain a D-configured aspartic acid or threonine residue. Combined, the
amatoxins and phallotoxins comprise 16 known compounds, and diversity among each class
arises from differences in both amino acid sequence and side-chain hydroxylation patterns [26].
6
Figure 1.3: Amatoxin and Phallotoxin Structure. α-Amanitin (A) and phalloidin (B)
are bicyclic peptides with cyclic backbones, side-chain hydroxylations (shown in red),
and a tryptophan-cysteine linkage (shown in blue).
Other cycloamanides that have been isolated include the virotoxins [33], cycloamanides A
through D [34], antamanide [35], and amanexitide [36] (Figure 1.4). Virotoxins are similar in
structure to the phallotoxins but are monocyclic and contain a tryptophan residue methylsulfonyl
modification. The other known cycloamanides, including antamanide and amanexitide, range
from six to ten amino acids in length and contain cyclic backbones and unmodified side-chains.
All of the post-translational modifications present among the amatoxins, phallotoxins, and other
cycloamanides are difficult or currently not possible to achieve in peptides using synthetic
chemistry, with the exception of incorporating D-amino acids into peptides. Therefore, insights
into cycloamanide biosynthesis and characterization of the enzymes involved may provide new
routes and synthetic strategies to these modifications.
7
Figure 1.4: Other Cycloamanide Compounds. Structures of cycloamanides A through
D (CyaA-D), antamanide (ANT), and viroisin (a virotoxin) are shown.
8
1.3 Ribosomal Biosynthesis of Cycloamanides
Although the amatoxins and phallotoxins were intially isolated in the 1930s and 1940s [26], no
details regarding their biosynthesis were known until 2007, when they were identified as
ribosomal peptides. Using a BLAST query for the amino acid sequences of α-amanitin
(IWGIGCNP) and the phallotoxin phallacidin (AWLVDCP), Hallen et al. (2007) [25] identified
sequences in the genome of the poisonous mushroom Amanita bisporigera that could encode the
toxins. The sequences were located within longer open reading frames with conserved upstream
and downstream sequences, and each encoding a translation product 34 or 35 amino acids in
length. The sequences were shown by Southern blot analysis to be present only in species of
mushrooms that produce these toxins (Figure 1.5) and were named AMA1 and PHA1 for α-
amanitin and phallacidin, respectively. Targeting conserved regions of the sequences, PCR and
additional BLAST searches revealed 14 additional sequences, all encoding predicted
oligopeptides beginning with the sequence ‘MSDIN.’ It was concluded that the amatoxins and
phallotoxins were products of a ribosomal biosynthesis pathway with a conserved ‘MSDIN’ gene
family encoding precursor peptides to the toxins and to the other cycloamanides.
The sequence structure of the cycloamanide precursor peptides is shown in Figure 1.6, as
revealed through a multiple sequence alignment of the MSDIN sequences identified in A.
bisporigera [37]. The sequences consist of conserved N-terminal (10mer) and C-terminal
(17mer) regions that flank an internal hypervariable sequence that contains the amino acids
found in the final cyclic peptide products. All known cycloamanides contain at least one proline
residue [26], and each internal sequence invariably starts and ends with proline. The degree of
conservation among the MSDIN sequences strongly implies that cycloamanide biosynthesis is
9
combinatorial, and that after translation the precursors function as ‘scaffolds,’ with recognition
sequences for the same core biochemical machinery and tailoring enzymes resulting in a variety
of cyclic peptides.
Figure 1.5: Southern Blot Analysis of Amanita Mushrooms. Lanes 1-4 contain
genomic DNA from α-amanitin and phallacidin producing species, and Lanes 5-13 from
non-toxic species. A, probed with AMA1 cDNA. B, probed with fragment of β-tubulin
gene. C, probed with PHA1 DNA. D, stained with ethidium bromide. Reprinted with
permission from Hallen et al., 2007 [25]. Copyright © 2007 National Academy of
Sciences.
Figure 1.6: Primary Structure of the Cycloamanide Precursor Peptides. The image
shows a WebLogo representation from a multiple sequence alignment of the 19 translated
MSDIN sequences identified in A. bisporigera, with the degree of conservation at each
position indicated by the height of each residue. The amino acid sequences of the
cycloamanides are located internally and are flanked by conserved N- and C-terminal
domains. Reprinted with permission from Luo et al., 2009 [44]. Copyright © 2009
ASBMB.
10
Species in the mushroom genus Galerina such as G. marginata are also known to produce
amatoxins [38,39,40]. Unlike A. bisporigera, which contains at least 19 MSDIN sequences, only
two genes (GmAma1-1 and GmAma1-2) are found in the G. marginata genome, both encoding
the α-amanitin sequence [41]. The precursor peptide sequences in Galerina diverge slightly from
those in Amanita, especially in the C-terminal domain, and Figure 1.7 shows an alignment of the
two AMA1 sequences. Although cycloamanide biosynthesis appears to be more limited in
Galerina, G. marginata may serve as an excellent model organism for characterization of the
pathway since its genome and transcriptome have been fully sequenced and annotated [41], and
unlike Amanita spp. it can be cultured in the laboratory [42,43].
Figure 1.7: Compared Sequences of Amanitin Precursors from Amanita and
Galerina. Sequences are from G. marginata (GmAMA1, top) and A. bisporigera
(AbAMA1, bottom). Divergent residues are highlighted in red and the internal α-amanitin
sequence is underlined.
While some of the genes identified in A. bisporigera contained sequences for known
cycloamanides, the majority are predicted to encode previously undiscovered cycloamanides and
new natural products. Therefore, a detailed understanding of cycloamanide biosynthesis may
provide a means to access new bioactive natural products, as well as characterized
cycloamanides that are present in mushrooms in low abundance.
11
WORKS CITED
12
WORKS CITED
1. Craik DJ. (2006). Seamless proteins tie up their loose ends. Science 311(5767): 1563-1564.
2. Laupacis A, Keown PA, Ulan RA, McKenzie N, and Stiller CR (1982). Cyclosporin A: a
powerful immunosuppressant. Can. Med. Assoc. J. 126(9): 1041-1046.
3. Du Vigneaud V, Ressler C, and Trippett S. (1953). The sequence of amino acids in
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16
CHAPTER 2
DETECTION AND PROFILING OF AMATOXINS IN
LEPIOTA MUSHROOMS
Note: The content in this chapter has been previously published. Some text has been modified
from the original. Copyright © 2014 by the authors; licensee MDPI, Basel, Switzerland.
Citation: Sgambelluri RM, Epis S, Sassera D, Luo H, Angelos ER, and Walton JD. (2014).
Profiling of amatoxins and phallotoxins in the genus Lepiota by liquid chromatography
combined with UV absorbance and mass spectrometry. Toxins. 6(8): 2336-2347.
Author Contributions: Fungal specimens were collected and identified by Sara Epis and Davide
Sassera. Evan R. Angelos and Hong Luo confirmed taxonomic identifications with ITS
sequencing.
17
2.1 Abstract
Ingestion of mushrooms in the genus Lepiota can result in fatal poisonings. Although clinical
symptoms and low resolution methods indicated that toxicity is due to the presence of amatoxins,
the toxin composition of Lepiota mushrooms has not been analyzed by modern high resolution
techniques. The spectrum of peptide toxins present in five species of Lepiota were analyzed by
liquid chromatography-mass spectrometry (LCMS). Field taxonomic identifications were
confirmed by sequencing of the internal transcribed spacer (ITS) regions. Extracts of other
poisonous mushrooms with previously characterized and well defined toxin profiles, including
Amanita phalloides, A. virosa, and Galerina marginata, were analyzed for comparison. The
compounds α-amanitin, β-amanitin, amanin, and amaninamide were detected in all isolates of L.
brunneoincarnata, and α-amanitin and γ-amanitin were detected in all isolates of L. josserandii.
Phallotoxins were not detected in either species. No amatoxins or phallotoxins were detected in
L. clypeolaria, L. cristata, or L. echinacea.
18
2.2 Introduction
The amatoxins, such as α-amanitin, are a group of bicyclic peptides produced by some species of
mushrooms and account for the majority of fatal mushroom poisonings worldwide [1]. They
display potent inhibition of eukaryotic RNA polymerase II, and factors that contribute to their
toxicity include resistance to heat and the digestive tract, and active intestinal and cellular uptake
[2]. Amatoxin poisoning is clinically manifested as symptoms of gastroenteritis resolving into an
asymptomatic period and ultimately followed by fulminant liver failure. In severe cases, liver
transplantation is the sole recourse [3]. In clinical settings, amatoxin poisoning is often assumed
on the basis of hepatic misfunction subsequent to mushroom ingestion, even in the absence of
chemical evidence [1,3].
Structurally, the amatoxins comprise the amino acid sequence Ile-Trp-Gly-Ile-Gly-Cys-Asn/Asp-
Pro, cyclized by head-to-tail peptide bonds and also a ‘tryptathionine’ side-chain linkage
between tryptophan and cysteine residues. Further diversity among of the amatoxins arises from
differences in hydroxylations of the side chains, which include 4-hydroxy-Pro, γ,δ-dihydroxy-Ile,
and 6-hydroxy-Trp (Figure 2.1). All of the amatoxins contain a cysteine with a sulfur oxidized
to the sulfoxide [4].
The phallotoxins, such as phalloidin and phallacidin, are a related class of bicyclic heptapeptides
that also contain tryptathionine. The phallotoxin core sequence is Ala-Trp-Leu-Ala/Val-D-
Asp/Thr-Cys-Pro, and differences in hydroxylations also generate structural diversity (Figure
2.1) [4]. Phallotoxins bind and stabilize F-actin, and their fluorescent conjugates are used as
cytological reagents to delineate the actin cytoskeleton [5].
19
Figure 2.1: Amatoxin and Phallotoxin Families of Bicyclic Peptides. Numbers in
parentheses after the compound names refer to the peak numbers in chromatography
traces shown later in the text.
Although species in the Amanita and Galerina genera (Figure 2.2A-C) are the most notorious
source of amatoxins and account for most fatal mushroom poisonings, numerous deaths have
also been attributed to ingestion of Lepiota, a genus of small, saprobic mushrooms distributed
worldwide (Figure 2.2D-H) [6-12]. However, in constrast to Amanita spp., there have been
relatively few analyses of the toxic composition of Lepiota and none using modern high
resolution methods. To date, chemical studies of Lepiota species have been restricted to thin
layer chromatography (TLC), which has poor resolution and relies on nonspecific visualization
reagents for identification, and the Meixner test. The Meixner test is a qualitative assay
developed in 1979 for amatoxins that involves blotting a sample onto paper and addition of
concentrated hydrochloric acid [13]. The formation of a blue color upon acid treatment is
indicative of amatoxins; however, the method suffers from a high rate of false positives from
20
reactions with other compounds such as substituted indoles, and is no longer considered a
reliable assay for amatoxin identification [14,15]. To redress the relative scarcity of information
regarding the distribution and abundance of amatoxins and phallotoxins in the clinically
significant Lepiota genus, five species of Lepiota were analyzed for toxin content by liquid
chromatography-mass spectrometry (LCMS).
Figure 2.2: Mushroom Species Analyzed in this Study for Toxin Content. A, Amanita
phalloides. B, A. virosa. C, Galerina marginata. D, Lepiota josserandii. All photographs
reprinted with permission from Mykoweb (http://www.mykoweb.com). Copyright ©
1996-2016, Michael Wood and Fred Stevens.
21
2.3 Methods
2.3.1 Mushroom Collection and Identification. Lepiota brunneoincarnata, L. clypeolaria, L.
cristata, L. echinacea, and L. josserandii mushrooms were collected in the Lombardy region of
Italy during the period of May 2012 through November 2013 by Sara Epis and Davide Sassera
(Department of Veterinary Sciences and Public Health, University of Milan), including multiple
isolates from different locations. For comparison, specimens of Amanita phalloides and A. virosa
were also collected from Italy and California, USA. All mushrooms were morphologically
identified by local expert mycologists with standard taxonomic keys. Galerina marginata was
obtained from Centraalbureau voor Schimmelcultures (CBS), Utrecht, Netherlands (catalog
number 339.88) and laboratory grown as described by Muraoka and Shinozawa (2000) [16]. All
specimens were freeze-dried or dried at room temperature and then stored at -80˚C.
Lepiota species identifications were confirmed by sequencing of the internal transcribed spacer
(ITS) regions. ITS regions were amplified using primer pairs ITS1 and ITS4 [17]. For template
preparation, approximately 1 mg of dried mushroom was homogenized with a tissue grinder in
50 μL of lysis buffer as described in Al Shahni et al. (2009) [18]. The samples were centrifuged
at 15,000 x g in a microfuge (Eppendorf 5415D) for 2 min and 1 μL of the supernatant was used
as the PCR template. PCR was performed under standard conditions using RedTaq polymerase
(Sigma, St. Louis, MO) in a total reaction volume of 20 μL. The DNA products of the reaction
were cloned into pGEM-T-Easy vector (Promega, Madison, WI) and sequenced by Sanger
technology. Sequences (Figure S2.1) were compared to nucleotide sequences in GenBank.
22
2.3.2 Toxin Extraction and LCMS. The dried fungal tissues were frozen in liquid nitrogen,
ground with a mortar and pestle, and suspended in methanol: H2O:0.01 M HCl, 5:4:1 at a
concentration of 10 mL/g tissue [19]. Following a one hour incubation at room temperature, the
extracts were centrifuged at 10,000 x g for 10 min, and the supernatants were filtered through a
0.22 μm filter (Millex polyvinylidene fluoride, GV4, Thermo Fisher Scientific, Waltham, MA).
Samples were stored at -80˚C until analysis. Immediately prior to HPLC fractionation, the
extracts were diluted with 20 mM ammonium acetate, pH 5, to a concentration of 20 mg dry
weight/mL.
The fungal extracts were separated on a reversed-phase Proto 300 C18 column (Higgins
Analytical; 5 μm, 250 x 4.6 mm) using an Agilent series 1200 HPLC equipped with a multi-
wavelength detector. Solvent A was 0.02 M ammonium acetate, pH 5, and solvent B was
acetonitrile. Toxins were separated with a stepwise gradient of 10% B for 4 min, 18% B for 6
min, and then a linear gradient from 18% B to 100% B over 20 min at a constant flow rate of 1
mL/min [19]. In each run, the equivalent of 0.6 mg of tissue was injected in a volume of 30 μL,
except for the G. marginata extract, for which 3 mg was injected. Mass analysis of the eluate
was performed with an Agilent 6120 single quadrupole mass spectrometer in positive ion mode.
Ions were generated by electrospray with a capillary voltage setting of 5 kV, a drying gas
(nitrogen) temperature of 350˚C, and flow rate of 12 L/min.
UV absorbance of the eluate was monitored at 280, 295, and 305 nm, because amatoxins and
phallotoxins exhibit an absorbance maximum (λmax) at 295 nm due to the presence of
tryptathionine, and the presence of 6-hydroxytryptophan shifts the λmax to 305 nm [4,20].
Quantitation of α-amanitin was based on absorbance at 305 nm and an external standard curve of
commercial α-amanitin (Sigma, St. Louis, MO).
23
2.4 Results
2.4.1 Toxins in Amanita and Galerina Mushrooms. The toxin profiles of Galerina marginata,
Amanita phalloides, and A. virosa are well characterized and remarkably consistent among
reported analyses [4,19,21,22]. Since no standards are commercially available for the majority of
amatoxins and phallotoxins, extracts of these mushrooms were analyzed as a benchmark and
source of standards for which mass, UV absorbance, and retention times could be compared.
G. marginata produces only α-amanitin, β-amanitin, and γ-amanitin in significant quantities
[19,23,24], and our extracts contained three prominent peaks with masses corresponding to these
compounds. A. phalloides is known to contain significant levels of α-amanitin, β-amanitin,
amanin, phallacidin, phallisacin, phallisin, and phalloidin. Using the same separation method as
Enjalbert et al. (1992) [19], all seven compounds were observed in A. phalloides extracts with
the expected elution order, nominal masses, and absorbance maxima of 305 nm for compounds
containing both tryptathionine and 6-hydroxytryptophan or 295 nm for compounds containing
only tryptathionine. As reported by Smith et al. (2012) [21], an apparent phallisin analogue
(referred to as ‘phallisin II’) with the same mass and UV absorbance as phallisin was also present
in the European A. phalloides isolate.
A. virosa is unique among other poisonous mushrooms in that it lacks β-amanitin and contains
amaninamide, which is structurally similar to α-amanitin but lacks 6-hydroxytryptophan [25].
Amaninamide was detected in the A. virosa extract along with α-amanitin, phallisin II,
phallacidin, and phalloidin. β-amanitin was absent as expected. Both Amanita species analyzed
contained several additional compounds (compounds 11 through 14) that are suspected to be
uncharacterized amatoxins or phallotoxins based on mass range and UV absorbance profiles.
24
2.4.2 Toxins in Lepiota Mushrooms. Lepiota brunneoincarnata and L. josserandii are common
Lepiota spp. associated with hospitalizations, and in agreement with the reported clincal features
and symptoms of ingestion, amatoxins were detected in both species. On the basis of mass, UV
absorbance, and column retention time, all isolates of L. brunneoincarnata contained α-amanitin
and β-amanitin, and the corresponding analogues lacking 6-hydroxytryptophan, amaninamide
and amanin. In L. josserandii, all isolates contained only α-amanitin and γ-amanitin. As in
Galerina, neither species contained phallotoxins. While no case reports identify them
specifically, L. clypeolaria, L. cristata, and L. echinacea are often listed as poisonous, however,
no amatoxins or phallotoxins were detected in all isolates of these species.
Table 2.1: Compounds Identified in Extracts of Amanita, Galerina, and Lepiota
Mushrooms. Compounds are numbered in order of elution time. Observed masses are
monoisotopic from singly charged ions.
Table 2.1 lists all of the compounds identified in these studies within mushroom extracts and the
toxin profiles are compared in Figure 2.3. While the levels of α-amanitin in L. brunneoincarnata
Peak Number Compound Expected Mass (Da) Observed Masses (m/z)1 β-amanitin 919.338182 920.3 [M+H], 942.4 [M+Na], 958.4 [M+K}
2 α-amanitin 918.354170 919.3 [M+H], 941.2 [M+Na], 957.2 [M+K}
3 amanin 903.343267 904.3 [M+H], 926.3 [M+Na], 942.2 [M+K}
4 phallisacin 865.316720 863.3 [M+H], 885.3 [M+Na], 901.2 [M+K}
5 γ-amanitin 902.359252 903.4 [M+H], 925.4 [M+Na], 941.3 [M+K}
6 phallisin II 804.311240 805.3 [M+H], 827.3 [M+Na], 843.2 [M+K}
7 amaninamide 902.359252 903.3 [M+H], 925.3 [M+Na], 941.2 [M+K}
8 phallacidin 846.321804 847.3 [M+H], 869.3 [M+Na], 885.3 [M+K}
9 phallisin 804.311240 805.4 [M+H], 827.3 [M+Na], 843.3 [M+K}
10 phalloidin 788.316330 789.3 [M+H], 811.3 [M+Na], 827.3 [M+K}
11 unknown x 789.2, 811.3, 827.2, 848.3, 889.3, 911.3, 927.2
12 unknown x 872.5 , 893.4, 914.5
13 unknown x 915.4, 937.4, 953.3, 960.6, 974.4
14 unknown x 755.3, 795.3, 811.2, 832.4, 869.5, 891.5
25
(on average, 0.76 mg per gram of dry tissue) were comparable to those in Amanita mushrooms,
the L. josserandii isolates contained an average of 4.2 mg α-amanitin per gram of dry weight,
which is more than three times higher than Amanita spp. and the highest reported levels of the
toxin to date (Table 2.2).
Figure 2.3: Toxin Profiles of Amanita, Galerina, and Lepiota Mushrooms. Signals are
overlaid UV absorbances at 295 nm (blue) and 305 nm (red). The identities and observed
masses for each peak are listed in Table 2.1. Peaks are labeled in order of retention time
and shared numbers between traces indicate the same compound. The shift in retention
time for compound 2 in the L. josserandii extract is due to column performance and is
within the deviations observed for standards.
26
Table 2.2: α-Amanitin Concentrations in Mushrooms. Concentrations were calculated
using absorbance at 305 nm and a standard curve of α-amanitin.
Species Isolate α-amanitin content (mg/g dry weight)
A. phalloides (Europe) 1 1.33
A. phalloides (USA) 1 0.88
A. virosa 1 1.39
G. marginata 1 0.57
L. brunneoincarnata 1 0.82
L. brunneoincarnata 2 0.69
L. josserandii 1 4.24
L. josserandii 2 4.39
L. josserandii 3 3.99
27
2.5 Discussion
This work details the first high-resolution analysis of cyclic peptide toxins in Lepiota
mushrooms. Structural identifications were made on the basis of mass, UV absorbance
(including diagnostic differences in λmax), and comparisons to extracts of other mushrooms with
well-defined toxin profiles. The results indicate that Lepiota brunneoincarnata and L. josserandii
produce amatoxins. Based on α-amanitin quantitation, L. josserandii is over three times more
toxic than Amanita species, and ingestion of a single fruiting body could prove fatal. No
amatoxins or phallotoxins were observed in extracts of L. clypeolaria, L. cristata, or L.
echinacea; however, Lepiota species can be difficult to identify without molecular tools, and
therefore none should be considered edible. Two additional Lepiota species, L. chlorophyllum
[9] and L. helveola [8], have been specified in case reports as the cause of mushroom poisoning.
While their toxin content remains to be determined, their toxicity is likely due to the presence of
amatoxins since these compounds have now been confirmed in other Lepiota species. Further
characterization and genomic studies of the amatoxin-producing Lepiota species identified in this
work may provide important insights into the biosynthesis of amatoxins and other cyclic peptides
in mushrooms.
28
APPENDIX
29
APPENDIX
Lepiota brunneoincarnata
TCCGTAGGTGAACCTGCGGAAGGATCATTATTGAATAAACTTGGTGGGTTGTTGCTGGCTTC
TTGGAGCATGTGCACGCTCATCGACTTTATCCATCCACCTGTGCACCTTCTGTAGTCTTCGAA
ATGAAAGCGGCTGAGCCTCGATGGGCATTTTGCCCTATCGGATGTGAGGAATGCTTTTGTGA
AGGCATGGCTCTCCTCAAAGGCCTGTGATCGTTTCTTGGACTATGTTTTTCCATATACCACAT
AGCATGTTGTAGAATGTATCGGTGGGCCTCTGTGCCTATAGAACTCAATACAACTTTCAGCA
ACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATT
GCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCTCCTTGGTATTCCGAGGAG
CATGCCTGTTTGAGTGTCATTTAATTCTCAACCATGCTGGCTTTGTAAAGGTCAGTTGTGGCT
TGGATTGTGGGGGTATTCCTGCGGGTCTCTCTTGAGGTCGGCTCCCCTAAAATGCATTAGCA
GAACCGTTTGCGGTCAGTCGCAGGTGTGATAATTATCTACGCCAAAGACCAAGGCTGCTCTC
TGTTTGTTCAGCTTCTAATTGTCTCGGGACAAATTTTTTTGAATGTTTGACCTCAAATCAGGT
AGGACTACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA
Lepiota clypeolaria (synonym L. magnispora)
TCCGTAGGTGAACCTGCGGAAGGATCATTATTGAATAACTATGGTGGGTTGTTGCTGGCTTC
TTGAAGCATGTGCACACCTGCTGTCTTTATCTATCCCACTGTGCACCATTTGTAGTCTTGGAG
GGGGAAGAGCGGTGAAGCTCACATGCCCCCCCTTCCGGGTCTATGTCTTTTCCACAAACATT
GTAGTATGTCACAGAATGTAATCAAAGGGTCTTTGTGCCCATAAAACTATATACAACTTTCA
GCAACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTG
AATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCTTCTTGGTATTCCGAG
GAGCATGCCTGTTTGAGTGTCATTAAATTCTCAATCCCTTCCAGTATTCTGGTTGTGGCTTGG
ATATTGGGGGTTTCTGCAGGCCTTATTATGTTGAGGTCAGCTCCCCTAAAATACATTAGCAG
AACTGTTTGCGGTCTGTCACTGGTGTGATAATTATCTGCACCAAGGCTGCTTTCTATCTTGTT
CAGCTTCCAACCGTCTTCTTGGAGACAACTATTGAACATTTGACCTCAAATCAGGTAGGACT
ACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA
Figure S2.1: ITS Sequences from Species of Lepiota Mushrooms.
30
Figure S2.1 (cont’d)
Lepiota cristata
TCCGTAGGTGAACCTGCGGAAGGATCATTATTGAATAAACTTGGTAGGTTGTAGCTGGCTTT
TCGAAGCATGTGCACGCCTACTATCTTTATCCATCCACCTGTGCACCCTTTGTAGTCTTGGAG
GACAAGAGCGGCTGACTCCTCGAACGGCTTCTTCTAGCCTTTCGGATGTGAGGGATGCTGTG
TGAAAGCACRGCTCTCCTCAATGGCTCGCAATTTCCTCTAGGTCTATGTCTTTTCCATATACC
ACATAGTATGTTGTAGAATGCATTATATGGGCCCATGTGCCTATAAAACTCAATACAACTTT
CAGCAACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATG
TGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCTCCTTGGTATTCCG
AGGAGCATGCCTGTTTGAGTGTCACTAAATTCTCAACCACTCCAGCCTTTGCGGGTTGGATG
TGGCTTGGATGTTGGGGGTTTCTGCGGGCCTCTCTTTTGAGGTCGGCTCCCCTGAAATGCATT
AGCGGAACCGTTTGCGGTCCGTCGCCGGTGTGATAATTATCTACGCCATAGACGAAGGCTGC
TCTCTGTATGTTCAGCTTCTAACTGTCCCCTGTGGACAACTTTTTGAACGTTTGACCTCAAAT
CAGGYAGGACTACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA
Lepiota echinacea
TCCGTAGGTGAACCTGCGGAAGGATCATTATTGAATAAACCTGGTGGGCTGTAGCTGGCTCT
TCGGAGCATGTGCACRCTCATCCACTTTTATCCATCCACCTGTGCACCATGTGTAGTCTTGGG
GGAGAAAGATTTGCGGTCCCGCTGTgGGCTTGTGAAGACGTCCTCTCAATTCTATGTTTTTCA
TATACCACRTAGTATGTTGCAGAATGTAtATAACGGGCCTATGTGCCTATAAAACACAATAC
AACTTTCAGCAACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAAG
TAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCTCCTTGGT
ATTCCGAGGAGCATGCCTGTTTGAGTGTCATTATATTCTCAACCCTTCCCAGTTWTAATGACT
TGGGTAAGTGGATTGGATTGTGGGGGCTTGCTGGTCGCTTTACTGCGGTCGGCTCCTCTGAA
ATGTATTAGCGGAACTGTTTGCGGTCcCGTCACTGGTGTGATAATTATCTACGCCGAAGACG
AAGGCTGCTCTCTATACGTTCAGCTTATAATCAGTCCCCTcTGGtGGACAACTTTTGAAAGTTT
GACCTCAAATCAGGTAGGACTACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA
Lepiota josserandii (synonym L. subincarnata)
TCCGTAGGTGAACCTGCGGAAGGATCATTATTGAATAAACATGGTGGGTTGTCGCTGGCTCC
TTGGAGCATGTGCACGCTCATCGTCTTTATCCATCCACCTGTGCACCTTTTGTAGTCTTGGGA
AATGAATGCAATGGAACCTCGATAGGTTTTTCAGCCTTTCGGATGTGAGGAATGCTTTGTGA
AAGCATGGCTCTTCTCAATAGCCTTGCAATCGTTACTCAGACTATGTTTTTCATACACCATGT
AGTATGTTTGCAGAATGTATCAATGGGCCTCTGTGCCTATAAAACTCAATACAACTTTCAGC
AACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAMGTAATGTGAA
TTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCTCCTTGGTATTCCGAGG
AGCATGCCTGTTTGAGTGTCATTTAATTCTCAACCACAAAGGCTTTTGCGAGCTTTTGTGGAT
TGGACGTGGGGGTAACTGCAGGCCTTCCCAGGTCAGCTCCCCTAAAATGCATTAGCGGAACC
GTTTGCGGTAACCAGTCGCCAGGTGTGATAATTATCTACGCCAATAGACATGAACTGCTCTC
TGTTGTTCTGCTTCAAATTGTCTTGCTAGACAACTTTTGAATGTTTGACCTCAAATCAGGTAG
GACTACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA
31
WORKS CITED
32
WORKS CITED
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14. Beutler JA, and Vergeer PP. (1980). Amatoxins in American Mushrooms: Evaluation of
the Meixner Test. Mycologia 72(6): 1142-1149.
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amanitin and false-positive reactions caused by psilocin and 5-substituted tryptamines.
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cultivation of the basidiomycete, Galerina fasciculata GF-060. J. Biosci. Bioeng. 89(1):
73-76.
17. White TJ, Bruns T, Lee S, and Taylor J. (1990). Amplification and direct sequencing of
fungal ribosomal RNA genes for phylogenies. In PCR Protocols: A Guide to Methods and
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CA, USA; pp. 315-322.
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to time-of-flight mass spectrometry separation for rapid assessment of toxins in
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23. Benedict RG, Tyler VE Jr, Brady LR, and Weber LJ. (1966). Fermentative production of
amanita toxins by a strain of Galerina marginata. J. Bacteriol. 91(3): 1380-1381.
24. Muraoka S, Fukamachi N, Mizomoto K, and Shinozawa T. (1999). Detection and
identification of amanitins in the wood-rotting fingi Galerina fasciculata and Galerina
helvoliceps. Appl. Environ. Microbiol. 65(9): 4207-4210.
25. Buku A, Wieland T, Bodenmuller H, and Faulstich H. (1980). Amaninamide, a new toxin
of Amanita virosa mushrooms. Experientia 36(1): 33-34.
34
CHAPTER 3
GENOMIC CAPACITY FOR CYCLOAMANIDE
BIOSYNTHESIS IN AMANITA MUSHROOMS
Note: The content in this chapter has been previously published. Some text has been modified
from the original. Copyright © 2016 by the authors.
Citation: Pulman JA, Childs KL, Sgambelluri RM, and Walton JD. (2016). Expansion and
diversification of the MSDIN family of cyclic peptide genes in the poisonous agarics Amanita
phalloides and A. bisporigera. BMC Genomics. 17(1): 1038.
Author Contributions: Assembly and annotation of fungal genomes and transcriptomes was done
by Jane A. Pulman and Kevin L. Childs. Field collection of fungal specimens and manual
annotation of MSDIN sequences was done by Jonathan D. Walton.
35
3.1 Abstract
Cycloamanides are cyclic peptide natural products found in Amanita, Galerina, and Lepiota
mushrooms and are produced from ribosomally-synthesized precursors. The precursor peptides
are encoded by the MSDIN gene family and are composed of conserved N- and C-terminal
domains and an internal hypervariable core domain containing the amino acids found in the final
peptides. While some MSDIN genes have been identified in genome surveys of Amanita species,
the full complement of MSDIN genes in a single species has yet to be reported. Draft genome
sequences were obtained for Amanita bisporigera and A. phalloides mushrooms and 31 MSDIN
genes were identified in the genome of A. bisporigera and 33 in A. phalloides, with a combined
total of 51 unique core domain sequences. RNAseq analysis of A. bisporigera confirmed
expression of 19 MSDIN sequences. Extracts of A. phalloides were searched for novel cyclic
peptides based on their expected masses and two new compounds, named cycloamanide E and
cycloamanide F, were demonstrated by LC/MS/MS. A. bisporigera and A. phalloides together
have the genetic capacity to synthesize at least 51 cycloamanides.
36
3.2 Introduction
The cycloamanide family of cyclic peptides produced by mushrooms includes the amatoxins,
phallotoxins, virotoxins, and other compounds including cycloamanides A through D (CyalA-D),
antamanide, and amanexitide [1,2,3,4]. Known bioactivities among the cycloamanides include
RNA polymerase II inhibition [5,6], actin binding and stabilization [7,8,9], immunosuppression
[10,11], and inhibition of the mammalian liver transporter OATP [12]. Cycloamanides are
biosynthesized from precursor peptides encoded by the MSIDN gene family. The precursors are
composed of a conserved N-terminal leader peptide domain, a hypervariable core region
containing the amino acid sequences of the cyclic peptides, and a conserved C-terminal domain
[13].
Discovery of the MSDIN gene family in genome surveys led to identification of 15 unique
MSDIN genes in Amanita bisporigera and 4 in other species, suggesting an extensive gene
family that gives rise to a large number of natural products [13]. Galerina mushrooms are also
known to produce amatoxins [14]; however, only two MSDIN sequences, both encoding α-
amanitin, were found in the complete genome of G. marginata [15]. The Amanita species A.
exitialis, A. fuliginea, A. fuligineoides, A. pallidorosea, A. phalloides, and A. rimosa have also
been searched for MSDINs by RNAseq [16] and PCR [17], and 42 MSDIN sequences with 28
unique core domains were found.
To date, a total of 36 unique MSDIN sequences have been identified and are predicted to encode
natural products. However, studies have been limited to incomplete genome and transcriptome
surveys and PCR, and therefore the full complement of MSDIN genes and capacity for cyclic
peptide biosynthesis has yet to be determined for a single species.
37
3.3 Methods
3.3.1 Genomics and Transcriptomics. Individual basidiocarps of Amanita bisporigera (Ab) and
A. phalloides (Ap) were collected in Ingham County, Michigan, in the summer of 2010, and in
Alameda County, California in the winter of 2011, respectively, by Jonathan D. Walton
(Department of Plant Biology and Department of Energy-Plant Research Laboratory, Michigan
State University). Genomic DNA and total RNA were isolated by organic solvent extraction
using cetyltrimethyl ammonium bromide (CTAB), phenol, and chloroform. DNA from each
species was sequenced using Illumina MiSeq technology, and RNA from Ab was reverse-
transcribed and sequenced using Illumina HiSeq. Sequencing was performed by the Michigan
State University RTSF Genomics Facility.
Assembly and annotation of the Ab and Ap genomes and Ab transcriptome was performed by
Jane A. Pulman and Kevin L. Childs (Department of Plant Biology and Center for Genomics-
Enabled Plant Science, Michigan State University). High-quality reads from Ab and Ap were
selected using Trimmomatic (ver 0.32) [18] and assembled using Velvet (ver 1.2.10) [19]. Gene
structural annotations were made using the MAKER pipeline [20,21] and functional annotations
using Trinotate (ver 2.0.2) [22]. MSDIN genes were identified by Jonathan D. Walton within
assemblies using tblastn with the conserved leader peptide sequence (MSDINATRLP) as query
and an e-value cutoff set to 100. Annotations of MSDIN genes were accomplished manually
with the aid of MAKER-predicted gene models and protein and transcript alignments with
known MSDIN genes.
3.3.2 LC/MS/MS of Predicted Cycloamanides. A lyophilized basidiocarp of Amanita
phalloides was ground to a powder in liquid nitrogen and resuspended in 90% ethanol at a
38
concentration of 1 g/50 mL. After stirring for 1 hr at room temperature, the ethanol was removed
under vacuum and the resulting residue was dissolved in a water/chloroform (1:1) solution. The
aqueous layer was collected and dried under vacuum and the residual oil was redissolved in 50%
acetonitrile.
This extract was analyzed using a Waters Xevo G2-XS QtoF HPLC/MS/MS system with a 5 uL
injection onto a BEH C18 UPLC column (2.1 mm x 50 mm, 1.7 µm particle size; Waters). The
column temperature was maintained at 30°C and the flow rate at 0.3 mL/min. Separation was
performed with 10 mM ammonium formate in water (solvent A) and acetonitrile (solvent B) with
an initial hold at 5% solvent B for 3 min followed by a linear gradient to 99% solvent B over 27
min. The MS settings were electrospray ionization (ESI) in positive mode, 3 kV capillary
voltage, 100°C source temperature, 350°C desolvation temperature, 600 L/hr desolvation
nitrogen gas flow, and 35 V cone voltage. Data were acquired using an MSe method having two
separate acquisition functions, where function 1 was performed with no collision energy and
function 2 was performed with a collision energy ramp from 60-100 V. For both functions, the
scan range was 50-1500 m/z with a scan rate of 0.2 seconds per function. Data were analyzed
using Masslynx (ver 4.1) (Waters) and mMass (ver 5.5.0) [23].
39
3.4 Results
3.4.1 MSDIN Genes in Amanita bisporigera and A. phalloides. The Ab genome contained
23,572 contigs assembled into 10,390 scaffolds and a total assembly size of 75 Mb with 74X
predicted fold coverage. The genome of Ap contained 5,437 contigs assembled into 1,465
scaffolds for an assembly size of 40 Mb and 69X predicted fold coverage. Identification of
MSDIN genes required tblastn searches and manual annotation, since none were annotated by
the MAKER tool even after the minimum length parameter was reduced to 150 base pairs. A
total of 64 MSDIN genes with 51 unique core domain sequences were identified in the Ab and
Ap genomes (Table 3.1). Ab contained 31 with 26 unique core domains and Ap contained 33
with 28 unique core domains. Expression at the level of RNA transcript was confirmed for 19 of
the unique sequences in Ab by RNAseq (Table S3.1). Only three core domain sequences were
common to both genomes, the α-amanitin (IWGIGCNP) and phalloidin (AWLVDCP) sequences
and ISDPTAYP. Of the 15 MSDIN sequences that were previously identified in genome surveys
of Ab [13], only 6 were present in our isolate. Similarly, genes encoding several cycloamanides
previously isolated from Ap (CylA, CylC, CylD and antamanide) [1] were absent in our Ap
isolate, suggesting significant intraspecies diversity in the gene family.
3.4.2 New Cycloamanides in Amanita phalloides. An extract of A. phalloides was searched for
new cycloamanides using extracted ion chromatograms for the predicted masses of the head-to-
tail cyclic, but otherwise unmodified peptides based on the genomic MSDIN sequences. Extracts
of Ap contained two compounds with masses corresponding to the cyclic versions of two
MSDIN core domain sequences, SFFFPVP and IVGILGLP. High resolution measurements
indicated a m/z of 822.4216 for putative cyclo-SFFFPVP (C45H56N7O8, calculated m/z 822.4190,
40
3.8 ppm error) and a m/z of 763.5118 for cyclo-IVGILGLP (C38H67N8O8, calculated m/z
763.5076, 5.5 ppm error). MS/MS confirmed the sequence of each compound, and by the
presence of unambiguous, overlapping fragments that span the entire sequence, the compounds
could be deduced as cyclic. The compounds were named cycloamanide E (SFFFPVP) (Figure
3.1) and cycloamanide F (IVGILGLP) (Figure 3.2).
Table 3.1: Cycloamanide Sequences in the Genomes of A. bisporigera and A.
phalloides.
A. bisporigera A. phalloides
AWLAECP AWLATCP
AWLVDCP AWLVDCP
CIGFLGIP FFFPPFFIPP
FFWPIIIPP FFPIVFSPP
FIWVLWLWLL FIFPPFIIPP
FNFFRFPYP FMPLAP
FSVLSIIPP FNILPFMLPP
GLGLIP FNLFRPYP
GLPIIAIIP GPVFFAY
GLPMVLP HFASFIPP
GMDPPSPMP IFLAFPIPP
GMEPPSPMP IFWFIYFP
IFWPIFAP IILAPIIP
IFWYIYFP IRLPPLFLPP
IGRPQLLP ISDPTAYP
IIFEPIIP IVGILGLP
ILMLAIPP IWGIGCDP
ISDPTAYP IWGIGCNP
IVFLEFYS LFFWFWFLWP
IWGIGCNP LGRPESLP
IWWYIYFP LILLAALGIP
LFFPPDFRPP LIQRPFAP
LFYPPDFRPP LPVLPIPLLP
LSSPMLLP LRLPPFMIPP
MAFPEFLA SFFFPIP
MIQRPFYP SFFFPVP
TIYYLYFIP
VQKPWSRP
41
Figure 3.1: MS/MS Analysis of Cycloamanide E. A, MS/MS spectrum. B, Peak list.
Peaks with more than one entry correspond to fragments with more than one possible
sequence. C, Overlapping fragments indicating a cyclic structure. The highlighted valine
is the same residue in each sequence.
Fragment #
M ………………......….V P S F F F P V
5 ……………..V P S F F
6 ……………..…..….P S F F
12 …………..…...….P S
9 …………..…….….P S F
2 …………..……….….P S F F P
10 ………………….………………...……...….F F
7 ……………...…...……………………….F F P
11 ………………..……………………………………...….F P
8 ………..…………………………………………...F P V
A
B C Peak Ion Meas. m/z Calc. m/z δ (Da) δ (ppm) Sequence
1 M 822.4216 822.4185 0.0031 3.8 SFFFPVP
2 b6 723.3519 723.3501 0.0018 2.5 PSFFFP
3 b6 675.3496 675.3501 -0.0005 -0.7 PVPSFF
3 b6 675.3496 675.3501 -0.0005 -0.7 FPVPSF
3 b6 675.3496 675.3501 -0.0005 -0.7 FFPVPS
4 b5 626.2978 626.2973 0.0005 0.8 SFFFP
4 b5 626.2978 626.2973 0.0005 0.8 PSFFF
5 b5 578.2992 578.2973 0.0019 3.3 VPSFF
6 b4 479.2286 479.2289 -0.0003 -0.6 PSFF
7 b3 392.1957 392.1969 -0.0012 -3.1 FFP
8 b3 344.1951 344.1969 -0.0018 -5.2 FPV
9 b3 332.1603 332.1605 -0.0002 -0.6 PSF
10 b2 295.1434 295.1441 -0.0007 -2.4 FF
10 b2 295.1434 295.1441 -0.0007 -2.4 FF
11 b2 245.1277 245.1285 -0.0008 -3.3 FP
12 b2 185.0903 185.0921 -0.0018 -9.7 PS
13 im4 120.0796 120.0808 -0.0012 -10.0 F
13 im3 120.0796 120.0808 -0.0012 -10.0 F
13 im2 120.0796 120.0808 -0.0012 -10.0 F
42
Figure 3.2: MS/MS Analysis of Cycloamanide F. A, MS/MS spectrum. B, Peak list.
Peaks with more than one entry correspond to fragments with more than one possible
sequence C, Overlapping fragments indicating a cyclic structure. The highlighted glycine
is the same residue in each sequence.
Fragment #
M………………......….G L P I V G I L G
9……………..G L P
4……………..…..….G L P I V G
8……………………………...…..…...….P I V
7……………………………………...…….….P I V G
6.………………………………………………………………………..….V G I L G
A
B
C Peak Ion Meas. m/z Calc. m/z δ (Da) δ (ppm) Sequence
1 M 763.5118 763.5076 0.0042 5.5 IVGILGLP
2 b7 650.4256 650.4236 0.002 3.1 PIVGILG
2 b7 650.4256 650.4236 0.002 3.1 GLPIVGI
2 b7 650.4256 650.4236 0.002 3.1 LGLPIVG
2 b7 650.4256 650.4236 0.002 3.1 VGILGLP
3 b6 593.4048 593.4021 0.0027 4.6 PIVGIL
3 b6 593.4048 593.4021 0.0027 4.6 LPIVGI
3 b6 593.4048 593.4021 0.0027 4.6 LGLPIV
4 b6 537.3364 537.3395 -0.0031 -5.8 GLPIVG
5 b5 480.3174 480.3180 -0.0006 -1.2 PIVGI
5 b5 480.3174 480.3180 -0.0006 -1.2 LPIVG
5 b5 480.3174 480.3180 -0.0006 -1.2 GLPIV
6 b5 440.2896 440.2867 0.0029 6.6 VGILG
7 b4 367.2352 367.2340 0.0012 3.3 PIVG
8 b3 310.2145 310.2125 0.002 6.4 PIV
9 b3 268.1665 268.1656 0.0009 3.4 GLP
10 b2 211.1453 211.1441 0.0012 5.7 PI
10 b2 211.1453 211.1441 0.0012 5.7 LP
43
3.5 Discussion
Besides Galerina marginata, which only contains two MSDIN sequences [15], this
chapter details the first complete assessment of MSDIN sequences in the genome of a
single species. Combined with previous studies in other species, a total of 73 MSDIN
sequences with unique core domains have been identified to date (Table S3.2). The core
domains range from 6 to 10 amino acids in length and all 20 amino acids are represented
at least once.
Because only two new cycloamanides were found in Ap extracts and not all were
expressed in Ab based on RNAseq data, it is possible that not all MSDIN sequences are
precursors to functional natural products. However, comparing the amino acid
distribution in the core domain sequences to expected frequencies based on the number of
codons for each residue reveals that the sequences are not random and that bulky
hydrophobic residues are highly overrepresented (Figure 3.3). This indicates a process
for genetic selection and suggests functionality in the products. Cycloamanides E and F
were identified in extracts as cyclic peptides with unmodified side-chains using predicted
masses based on genomic sequence, and other products of the pathway likely eluded
detection due to the presence of additional post-translational modifications. Future
studies should be aimed at describing these other post-translational modifications,
identifying new cycloamanides and their bioactivities, and describing the genetic
mechanisms behind the extensive duplication of the MSDIN genes and hypermutation of
their core domains.
44
Figure 3.3: Amino Acid Frequencies in MSDIN Core Domain Sequences. Values are
observed frequency minus expected frequency (%) for each amino acid, colored by type.
45
APPENDIX
46
APPENDIX
Table S3.1: List of Core Domains Identified Among MSDIN Transcripts from RNAseq of
Amanita bisporigera.
AWLAECP
AWLVDCP
FNFFRFPYP
FSVLSIIPP
GLPIIAIIP
GMDPPSPMP
GMEPPSPMP
IFWPIFAP
IFWYIYFP
IGRPQLLP
IIFEPIIP
ILMLAIPP
ISDPTAYP
IVFLEFYS
IWGIGCNP
IWWYIYFP
LSSPMLLP
MAFPEFLA
MIQRPFYP
47
Table S3.2: Alphabetical List of All Unique MSDIN Core Sequences Identified to Date.
Core Sequence Species
AWLAECP bisporigera, rimosa
AWLALCP fuligineoides
AWLATCP ocreata, phalloides
AWLTDCP exitialis
AWLVDCP bisporigera, exitialis, pallidorosea, phalloides
CIGFLGIP bisporigera
FFFPPFFIPP phalloides
FFPIVFSPP phalloides
FFQPPEFRPP bisporigera
FFWPIIIPP bisporigera
FIFPPFIIPP phalloides
FIWVLWLWLL bisporigera
FLFPPVRLPP bisporigera
FMPLAP phalloides
FNFFRFPYP bisporigera
FNILPFMLPP phalloides
FNLFRFPYP phalloides
FSVLSIIPP bisporigera
FVFVASPP exitialis
FYQFPDFKYP bisporigera
GAYPPVPMP bisporigera
GFVPILFP bisporigera
GLGLIP bisporigera
GLPIIAIIP bisporigera
GLPMVLP bisporigera
GMDPPSPMP bisporigera
GMEPPSPMP bisporigera
GPVFFAY phalloides
HFASFIPP phalloides
HLVRYPP fuligineoides
HPFPLGLQP bisporigera
IFLAFPIPP phalloides
IFWFIYFP exitialis, phalloides
IFWPIFAP bisporigera
IGRPQLLP bisporigera
IIFEPIIP bisporigera
IIGILLPP phalloides
IIIVLGLIIP rimosa
IILAPIIP phalloides
IIWAPVVP exitialis, fuliginea
48
Table S3.2 (cont’d)
Core Sequence Species
ILMLAILP bisporigera
ILMLAIPP bisporigera
IPGLIPLGIP bisporigera
IRLPPLFLPP phalloides
ISDPTAYP bisporigera, phalloides
IVFLEFYS bisporigera
IVGILGLP phalloides
IWGIGCDP exitialis, fuliginea, fuligineoides, pallidorosea, phalloides, rimosa
IWGIGCNP bisporigera, exitialis, fuliginea, fuligineoides, pallidorosea, phalloides, rimosa
IWWYIYFP bisporigera
LFFPPDFRPP bisporigera, exitialis
LFFWFWFLWP phalloides
LFLPPVRMPP bisporigera
LFYPPDFRPP bisporigera
LGRPESLP phalloides
LGRPFAP phalloides
LILLAALGIP phalloides
LIQRPFAP phalloides
LLILSILP exitialis
LPVLPIPLLP phalloides
LRLPPFMIPP phalloides
LSSPMLLP bisporigera
MAFPEFLA bisporigera
MIQRPFYP bisporigera
SFFFPIP phalloides
SFFFPVP phalloides
TIYYLYFIP phalloides
VFSLPVFFP exitialis
VQKPWSRP phalloides
VWIGCSP fuliginea
VWIGYSP exitialis, fuligineoides
WLATCP phalloides
YVVFMSFIPP bisporigera
49
WORKS CITED
50
WORKS CITED
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14. Enjalbert F, Cassanas G, Rapior S, Renault C, and Chaumont JP. (2004). Amatoxins in
wood-rotting Galerina marginata. Mycologia 96(4): 720-729.
15. Luo H, Hallen-Adams HE, Scott-Craig JS, and Walton JD. (2012). Ribosomal biosynthesis
of α-amanitin in Galerina marginata. Fungal Genet. Biol. 49(2): 123-129.
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transcriptome sequencing and analysis of Amanita exitialis basidiocarps. Gene 532(1):
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cyclic peptide tandem mass spectra. PLoS One. 7(9): e44913
52
CHAPTER 4
CHARACTERIZATION OF AMANITIN BIOSYNTHESIS
IN GALERINA MARGINATA
53
4.1 Abstract
Galerina marginata is a saprobic mushroom that produces the ribosomal bicyclic peptide toxin
α-amanitin. Unlike most basidiomycetes, G. marginata is culturable and thus may be useful as a
model organism for studying the biosynthesis of amanitin and related compounds. α-Amanitin
levels were quantified over time in laboratory grown G. marginata mycelium. On average,
amanitin production began after 25 days of growth and peaked after 40 days to 1.39 mg per gram
of dry tissue. Candidate biosynthetic genes were identified in the G. marginata genome based on
genome clustering with the gene encoding the amanitin precursor peptide, GmAMA1, and
included a predicted prolyl oligopeptidase (GmPOPB), flavin-containing monooxygenase
(GmFMO), and P450 monooxygenase (GmP450-29). G. marginata strains harboring knockouts
for the three candidates were developed and the effects on α-amanitin production were assessed
by HPLC. Production of the toxin was abolished in all three mutants, suggesting the involvement
of these enzymes in the pathway. In the P450-29(-) strain, an intermediate to α-amanitin
accumulated that had a mass corresponding to α-amanitin missing two hydroxylations of the
amino acid side-chains. NMR spectroscopy of the purified intermediate indicated the absence of
hydroxyl groups at the δ-position of Ile1 and the γ-position of Pro8. Expression patterns of the
genes known or hypothesized to be involved in the pathway were characterized by RT-PCR as a
potential avenue for identifying additional biosynthetic genes by patterns of co-expression.
Transcriptional activation of GmAMA1 correlated with the onset of toxin biosynthesis but no
correlation with expression of the other biosynthetic genes was observed.
54
4.2 Introduction
Cycloamanides such as amatoxins and phallotoxins are known to be synthesized by mushrooms
in the Amanita [1], Galerina [2], and Lepiota [3] genera. Culturing of higher fungi in the
laboratory is often difficult or unsuccessful due to the complex and poorly understood growth
requirements of these organisms. This growth issue remains true for the majority of
cycloamanide producers with the exception of Galerina marginata, a saprobic wood-rotting
mushroom that is distributed worldwide [4] (Figure 4.1). The ability to culture G. marginata [5]
makes this species a potentially useful model organism for studying cycloamanide biosynthesis.
The G. marginata genome and transcriptome have previously been fully sequenced and
annotated [6] (publicly available at http://jgi.doe.gov) . Unlike Amanita spp., G. marginata is
more limited in cycloamanide biosynthesis and only contains two MSDIN genes, GmAMA1-1
and GmAMA1-2, both of which encode the precursor peptide for α-amanitin. The precursor
peptides in G. marginata share the same overall structure as those from Amanita, with conserved
Figure 4.1: Basidiocarps of Galerina marginata. Reprinted with permission from
MykoWeb (http://www.mykoweb.com). Copyright © 1996-2016, Michael Wood.
55
leader and follower peptides and invariable proline residues flanking a core domain, although the
leader and follower sequences diverge significantly between Amanita and Galerina (see Figure
1.6).
Starting from a 35mer precursor peptide, steps in the biosynthesis of α-amanitin must include
proteolysis, four side-chain hydroxylations, a sulfoxidation, tryptathionine formation, and
backbone condensation/cyclization (Figure 4.2). While genes in pathways of secondary
metabolism are often clustered in fungi [7,8], no conserved cluster is apparent for the
cycloamanides. However, each MSDIN in G. marginata is found in close proximity to genes
with predicted functions that could be relevant to the pathway. GmAMA1-1 lies just downstream
of three genes predicted to encode P450 monooxygenases and adjacent to a predicted prolyl
oligopeptidase (POP) and flavin-containing monooxygenase (FMO). GmAMA1-2 also lies
adjacent to a predicted P450 monooxygenase (Figure 4.3). The putative monooxygenase
enzymes may be responsible for the side-chain hydroxylations seen in α-amanitin. The precursor
peptides contain conserved proline residues, and POP enzymes, which hydrolyze peptides at
prolines, are likely involved in their processing (see Chapter 5). In addition, two POP genes,
POPA and POPB are present in all mushroom species with available genomic data that produce
cycloamanides, whereas non-producers only contain POPA [6]. This finding suggests that POPB
might play a dedicated role in cycloamanide biosynthesis. The following studies aim to
characterize amanitin biosynthesis, including identification of the genes involved in laboratory
grown G. marginata.
56
Figure 4.2: Steps in the Biosynthesis of α-Amanitin from the GmAMA1 Precursor
Peptide. The order in which the tailoring steps occur is unknown.
Figure 4.3: Genes Adjacent to GmAMA1 in the G. marginata Genome with Relevant
Predicted Functions.
57
4.3 Methods
4.3.1 Galerina Growth and Toxin Analysis. G. marginata was obtained from Centraalbureau
voor Schimmelcultures (CBS), Utrecht, Netherlands (catalog number 339.88) and maintained on
potato dextrose agar. For α-amanitin production, a 1x1 cm square of new growth from a potato
dextrose agar (PDA) plate was used to inoculate 100 mL of “HSV-5C” medium [5] in a 250 mL
flask, and the mycelium was grown with shaking at room temperature. The growth medium
contained (per liter) 5 g glucose, 1 g yeast extract, 100 mg NH4Cl, 100 mg KCl, 100 mg
CaSO4•2H2O, 1 mg thiamine, and 0.1 mg biotin. The pH of the medium of was adjusted to 5.2
and then autoclaved. The mycelium was harvested from each flask in 1 or 2 day intervals by
filtering the culture through miracloth. The mycelium was then frozen and lyophilized, and the
dry weight was measured and recorded before storing at -80°C. A total of 17 time points were
collected, each in triplicate between 10 days and 50 days of growth.
For toxin extraction, the frozen samples were ground in a mortar and pestle, dissolved in
methanol:water:0.01 M HCl (5:4:1) at a concentration of 10 mL per gram of tissue, and
incubated for 1 hour at room temperature. The extracts were then centrifuged at 10,000 x g for
10 min and the supernatants passed through a 0.22 µm syringe filter. α-Amanitin was quantified
by HPLC and an external standard curve of commercial toxin (Sigma-Aldrich). Separation was
performed using an Agilent 1200 series HPLC with a multi-wavelength detector and a Proto 300
C18 reverse-phase column (Higgins; 5 µm, 250 x 4.6 mm). Solvent A was 0.02 M ammonium
acetate (pH 5) and solvent B was acetonitrile. The HPLC program used was developed by
Enjalbert et al., 1992 [9] and was 10% solvent B for 10 min, step to 18% solvent B for 6 min,
and then a linear gradient from 18% to 100% solvent B over 20 min at 1 mL/min. For each
58
sample, an equivalent of 3 mg of tissue was analyzed and the area of the absorbance peak
corresponding to α-amanitin (based on retention time and UV profile compared to standard and
confirmed with ESI-LCMS) was measured at 305 nm.
4.3.2 Galerina Transformation and Gene Knockouts. Targeted gene knockouts in
G.marginata were accomplished by Hong Luo (MSU-DOE Plant Research Laboratory,
Michigan State University) using an Agrobacterium tumefaciens mediated transformation
method developed for the mushroom Laccaria bicolor [10,11]. For each knockout, the T-DNA
cassettes contained a hygromycin resistance gene (hph, hygromycin B phosphotransferase) for
selection and 1.5 to 1.8 kbp of upstream and downstream genomic sequences for targeted
homologous recombination. Knockouts were confirmed in all transformants by PCR and
Southern blotting, and the effects on α-amanitin production were assessed in extracts of the
transformants by the HPLC method described in section 4.3.1.
4.3.3 Purification of an Amanitin Intermediate and NMR. A pathway intermediate to α-
amanitin was identified and purified from approximately 3 g (dried) of a G. marginata strain
harboring a knockout of a gene predicted to encode a P450 enzyme. The intermediate was
purified by reversed-phase HPLC in two steps on a semi-preparative C18 column (25 cm x 10
mm, 5 mm, Supelcosil LC-18). For the first separation, solvent A was 20 mM aqueous
ammonium acetate:acetonitrile (90:10, v/v) and solution B was 20 mM ammonium
acetate:acetonitrile (76:24, v/v), both adjusted to pH 5 with glacial acetic acid. A step-wise
gradient profile was used and consisted of 100% A for 3 min, 43% A for 7 min, and 0% A for 9
min at a constant flow rate of 2 mL/min. The second purification step consisted of a linear
gradient of 100% 20 mM ammonium acetate to 100% acetonitrile over 15 min. Both separations
59
were carried out on an Agilent 1200 series HPLC with a multi-wavelength detector. Fractions
containing the intermediate were pooled and lyophilized.
For NMR experiments, the intermediate was dissolved in DMSO-d6 to a final concentration of
3.8 mM. Spectra were recorded at 25˚C on a Bruker Avance 900 MHz instrument (Max T.
Rogers NMR Facility, Michigan State University) with a TCI cryoprobe. 2D DQF-COSY,
TOCSY, ROESY, 1H-
13C HSQC, and
1H-
13C HMBC experiments were performed for
assignment and structure determination using standard parameters. DMSO solvent was used as
the chemical shift reference and spectra of commercial α-amanitin (Sigma-Aldrich) were
recorded for comparison.
4.3.4 Analysis of Gene Expression by RT-PCR. Expression of genes involved in α-amanitin
biosynthesis, including GmAMA1-1, GmPOPB, and GmFMO, was analyzed by reverse
transcription-PCR (RT-PCR) before and after the onset of toxin production. Two β-tubulin genes
(GmTUBB1 and GmTUBB2) and the gene encoding POPA were also analyzed as controls. Total
RNA was prepared from G. marginata after 10, 20, and 40 days of growth using a RNeasy Plant
Kit (Qiagen), and the quality of the RNA was confirmed on an agarose gel by the presence of
intact 16S and 23S rRNA bands. cDNA was then synthesized using SuperScript III reverse
transcriptase (ThermoFisher) and an oligo-dT primer. PCR was performed with RedTaq
Polymerase (Sigma-Aldrich) with primers designed for the genes listed above based on the
available genomic and transcript sequences. Amplification of the correct target sequences was
confirmed by Sanger sequencing of the PCR products.
60
4.4 Results
4.4.1 Time Course of Amanitin Production. Production of the toxin α-amanitin by Galerina
marginata in laboratory cultures was measured in extracts of fungal tissue between 10 and 50
days of growth (Figure 4.4 and Figure 4.5). α-Amanitin production typically began after 20 to
25 days and peaked on day 40 at an average level of 1.39 mg per gram of tissue. This growth
duration was used in all subsequent experiments assessing the effects of knockouts of candidate
biosynthetic genes, since the difference between abolished versus diminished toxin production
phenotypes would be more apparent at higher levels. After 40 days, α-amanitin levels began to
decrease and fell to an average of 0.37 mg/g by day 50. No α-amanitin was detectable in the
media of these cultures, ruling out secretion of the toxin. Although α-amanitin is highly stable
and resistant to proteases [1], the observed disappearence of the toxin is likely the result of
turnover and catabolism by the host.
Figure 4.4: Culture of Galerina marginata Mycelium. Photo was taken after 20 days of
growth.
61
Figure 4.5: Time Course of α-Amanitin Production in Galerina marginata Cultures.
Overlaid plots of G. marginata dry biomass produced in grams (Blue) and amount of α-
amanitin per gram of tissue (Red). Error bars represent the range in toxin levels from
measurements of three separate cultures.
4.4.2 Genes Involved in Amanitin Biosynthesis. G. marginata strains harboring knockouts of
genes encoding predicted POPB, FMO, and P450 enzymes on the same scaffold as GmAMA1-1
were successfully engineered by Hong Luo (MSU-DOE Plant Research Laboratory, Michigan
State University). A knockout of the gene encoding a P450 adjacent to GmAMA1-2 was
unsuccessful, but a knockout of a predicted P450-encoding gene separated from GmAMA1-1 by
29 coding sequences (designated GmP450-29) was achieved. The effects on amanitin production
were assessed by LCMS from cultures of the strains grown alongside wild-type Galerina for 40
days, where toxin levels peak in the wild-type strain. No detectable levels of α-amanitin or any
of the less hydroxylated forms were present in extracts of the POPB(-) and FMO(-) mutants
(Figures 4.6 and 4.7). This result, in combination with their close proximity to GmAMA1-1 and
62
predicted functions, establishes their involvement in the pathway. The P450-29(-) mutant also
lost the capacity to produce α-amanitin (Figure 4.8), but we observed formation of a new peak in
extracts of the mutant that was absent from the wild-type and hypothesized it to be an
intermediate to α-amanitin. UV absorbance spectra of amatoxins and phallotoxins contain a peak
at 295 nm due to the presence of tryptathionine, and in compounds containing 6-
hydroxytryptophan this peak is shifted to 305 nm [1,3]. The suspected intermediate displayed
stronger absorbance at 305 nm versus 280 nm, suggesting the presence of both tryptathionine
and a modified tryptophan. In agreement with the disrupted gene’s predicted fuction as a
monooxygenase, the compound also displayed a mass of 886.4 m/z, 32 mass units less than α-
amanitin and consistent with an intermediate missing two of the four possible hydroxylations
that occur in α-amanitin.
Figure 4.6: POPB Knockout in Galerina and Effects on α-Amanitin Production.
Signals show UV absorbance (305 nm) of toxin extracts from both wild-type (blue) and
the mutant (orange) overlaid.
63
Figure 4.7: FMO Knockout in Galerina and Effects on α-Amanitin Production.
Signals show UV absorbance (305 nm) of toxin extracts from both wild-type (blue) and
the mutant (green) overlaid.
Figure 4.8: P450 Knockout in Galerina and Effects on α-Amanitin Production.
Signals show UV absorbance (305 nm) of toxin extracts from both wild-type (blue) and
the mutant (red) overlaid.
64
4.4.3 Role of a P450 Monooxygenase in Amanitin Biosynthesis. Approximately 0.7 mg of the
intermediate that accumulated in the P450-29(-) Galerina mutant was purified and dissolved in
DMSO-d6 for structure determination by NMR spectroscopy. The proton spectrum of the
intermediate was similar to that of α-amanitin (Figure S4.1), suggesting an overall related
structure. All correlations observed in 2D experiments (Figures S4.2-S4.7 and Table S4.1) were
consistent with the structure of a previously undescribed amatoxin shown in Figure 4.9, with
missing hydroxylations at the δ-position of Ile1 and γ-position of Pro8. Backbone HN-CO
correlations and interresidue NOEs in the intermediate indicated a cyclic backbone. Consistent
with γ-hydroxylation, only one Hγ proton was assigned to Ile1 and was coupled to a δ-methyl
group in the COSY experiment. HSQC with multiplicity-editing also indicated the absence of
CH2 groups in Ile1 (Figure 4.10), and the residue was therefore concluded to be unmodified at
the δ-position. For Pro8, all seven protons could be
Figure 4.9: Compared Structures of α-Amanitin and the Pathway Intermediate
Purified from a P450(-) Strain of G. marginata. Hydroxlyations are highlighted in red
and those missing from the intermediate are circled.
65
assigned (Figure 4.10), including γCH2 which was absent in α-amanitin. For both α-amanitin
and the intermediate, no couplings were observed with the side-chain indole NH of tryptophan,
and only three aromatic 1H-
13C bonds were present, consistent with tryptophan modified at
positions 2 (tryptathionine) and 6 (hydroxylation). The P450 enzyme is therefore proposed to be
responsible for δ-hydroxylation of isoleucine and/or γ-hydroxylation of proline in α-amanitin
biosynthesis.
Figure 4.10: Compared 1H-
13C HSQC Spectra of α-Amanitin and the Intermediate.
Signals assigned to the δ-position of Ile1 and the γ-position of Pro8 are indicated.
66
4.4.4 Regulation of Biosynthetic Genes. While the POP and monooxygenase enzymes are
likely involved in processing of the precursor peptide and hydroxylations, no candidate enzymes
have been designated for formation of the unique tryptathionine group seen in the amatoxins. In
fungi, biosynthetic genes for many secondary metabolites are transcriptionally co-regulated,
sometimes by a single transcriptional activator dedicated to the pathway [12,13]. If
transcriptional co-regulation occurs in the pathway for amanitin biosynthesis, then expression
profiling and microarray analysis may be an effective approach for identifying the remaining
biosynthetic genes. Expression of GmAMA1-1, GmFMO, and GmPOPB was analyzed before
(10 day culture) and after (25 day and 45 day cultures) the onset of α-amanitin production in G.
marginata (Figure 4.11) by reverse transcription-PCR. Only GmAMA1-1 expression correlated
with biosynthesis, with transcripts only detectable after 25 days and after the start of α-amanitin
production. GmFMO and GmPOPB showed constitutive expression along with genes encoding
β-tubulin and POPA. Transcriptional activation of GmAMA1-1 is likely limiting to the overall
pathway, and other approaches will be necessary for identifying the other remaining biosynthetic
genes.
67
Figure 4.11: Expression of Genes Involved in Amanitin Biosynthesis. Transcripts
were amplified from mRNA by RT-PCR from G. marginata mycelium grown 10 days
(before the onset of toxin biosynthesis), and after 25 and 45 days (during toxin
biosynthesis). AMA1-1 expression correlates with toxin production while POPB and
FMO show constitutive expression along with housekeeping genes encoding β-tubulin
and POPA.
68
4.5 Discussion
Three enzymes involved in the biosynthesis of α-amanitin, and possibly cycloamanides in
general, were identified in these studies. A predicted prolyl oligopeptidase (GmPOPB), a flavin-
containing monooxygenase (GmFMO), and a P450 monooxygenase (GmP450-29) that was
further shown to function in isoleucine and/or proline hydroxylation. It is unclear whether the
P450 is bifunctional and responsible for both hydroxylations, or catalyzes one hydroxylation to
provide a suitable substrate for a separate enzyme. Because no informative pathway
intermediates accumulated in the POPB(-) and FMO(-) mutants, the predicted functions of these
enzymes were originally based on bioinformatics and automated gene functional annotations.
Recombinant POPB has since been shown to encode the macrocyclase that converts the
precursor peptide to the cyclic intermediate [14] (see Chapter 5), and determining the precise
roles of the P450 and FMO enzymes will similarly require recombinant expression and
biochemical characterization.
The backbone and side-chain to side-chain (tryptathionine) cyclizations seen in α-amanitin are
largely uncharacterized modifications in natural products. At the time of this work, one enzyme,
PatG, responsible for N- to C-terminal cyclization in the biosynthesis of the cyanobactin family
of cyclic peptides, had been characterized [15]. PatG was determined to be a serine protease-
related enzyme catalyzing peptide bond ligation instead of hydrolysis, and a peptidase such as
POPB may share a similar function. Similarly, while the Trp-Cys linkage seen in amatoxins and
other cycloamanides is unique to these compounds and the responsible enzyme is unknown, the
FMO enzyme may also function in cyclization since successful synthetic routes to tryptathionine
have employed tryptophan with a hydroxylated indole that activates the side-chain for thiol
69
addition [16]. The accumulation of the intermediate resulting from the P450 knockout and
detectable presence of the other less modified versions of α-amanitin in wild-type Galerina such
as γ-amanitin [3] is indicative of the stability of these less hydroxylated forms, and the absence
of any accumulated intermediates or relevant compounds in the FMO(-) mutant suggests a
function more integral to the structure of the amatoxins such as cyclization.
70
APPENDIX
71
APPENDIX
v
Figure S4.1: 1H Spectra of α-Amanitin and the Pathway Intermediate Purified from a
P450(-) Strain of G. marginata.
72
Figure S4.2: 2D COSY spectrum of the Amanitin Intermediate.
Figure S4.3: 2D TOCSY spectrum of the Amanitin Intermediate.
73
Figure S4.4: 2D ROESY spectrum of the Amanitin Intermediate.
Figure S4.5: 2D 1H-
13C HSQC spectrum of the Amanitin Intermediate.
74
Figure S4.6: 2D 1H-
13C HMBC spectrum of the Amanitin Intermediate.
75
Figure S4.7: Key HMBC Correlations Observed in the Amanitin Intermediate for
Structure Determination.
76
Table S4.1: Table of all 2D NMR Correlations Observed in the Amanitin Intermediate. All
nuclei are listed numerically and for each, the couplings observed in each experiment are
indicated with numbers indicating the other coupled nuclei.
Residue Nucleus Atom ppm TOCSY COSY HSQC HMBC ROESY
1 Ile1 H HN 7.86 1,2,3,4,5,6 1,2 x 83 2,4,6,13,74
2 Ile1 H Hα 4.20 1,2,3,4,5,6 1,2,3 7 8,9,10,12 1,4,6,13
3 Ile1 H Hβ 1.99 1,2,3,4,5,6 2,3,5 8 7,9,10,11,12 x
4 Ile1 H Hγ 3.69 1,2,3,4,5,6 4,6 9 7,8,10,11 1,2,6,74
5 Ile1 H y'CH3 0.82 1,2,3,4,5 3,5 10 7,8,9 x
6 Ile1 H δCH3 0.92 2,3,4,6 4,6 11 8,9 1,2,4
7 Ile1 C Cα 55.87 x x 2 3,4,5 x
8 Ile1 C Cβ 40.97 x x 3 2,4,5,6 x
9 Ile1 C Cγ 65.41 x x 4 2,3,5,6 x
10 Ile1 C Cγ' 10.73 x x 5 2,3,4 x
11 Ile1 C Cδ 17.77 x x 6 3,4 x
12 Ile1 C CO 170.58 x x x 2,3,13,14 x
13 Trp2 H HN 8.04 13,14,15,16 13,14 x 12,21,22 1,2,14,15,16
14 Trp2 H Hα 4.81 13,14,15,16 13,14,15,16 21 12,22,31 13,15,18,32
15 Trp2 H Hβ1 3.26 13,14,15,16 14,15,16 22 21,23,24,25 13,14,16,18,67
16 Trp2 H Hβ2 2.68 13,14,15,16 14,15,16 22 21,23,24,25 13,15,32,62,67
17 Trp2 H 1(N)H 11.27 x x x x x
18 Trp2 H 4H 7.45 18,19,20 18,19 26 24,25,28,29,30 14,15,19
19 Trp2 H 5H 6.58 18,19,20 18,19 27 25,28,29 18
20 Trp2 H 7H 6.74 18,19,20 x 29 25,27,28,30 x
21 Trp2 C Cα 53.03 x x 14 13,15,16 x
22 Trp2 C Cβ 28.04 x x 15,16 13,14 x
23 Trp2 C C2 129.86 x x x 15,16 x
24 Trp2 C C3 111.28 x x x 15,16,18 x
25 Trp2 C C3a 120.66 x x x 15,16,18,19,20 x
26 Trp2 C C4 122.20 x x 18 x x
27 Trp2 C C5 110.41 x x 19 20 x
28 Trp2 C C6 154.66 x x x 18,19,20 x
29 Trp2 C C7 96.45 x x 20 18,19 x
30 Trp2 C C7a 138.76 x x x 18,20 x
31 Trp2 C CO 170.34 x x x 14,32 x
32 Gly3 H HN 7.88 32,33,34 32,33 x 31 14,16,33,34,57,62
33 Gly3 H Hα1 4.37 32,33,34 32,33,34 35 36 32,34,37
34 Gly3 H Hα2 3.27 32,33,34 33,34 35 36 32,33,37
35 Gly3 C Cα 40.82 x x 33,34 x x
36 Gly3 C CO 170.35 x x x 33,34,37 x
37 Ile4 H HN 8.45 37,38,39,41,42 37,38 x 36,45 33,34,39,41,42
38 Ile4 H Hα 3.60 37,38,39,41,42,43 37,38,39 44 45,47,49 x
39 Ile4 H Hβ 1.55 37,38,39,41,42,43 38,39,40,41,42 45 44,46,47,49 37
40 Ile4 H Hγ1 1.53 x 39,40,41,43 46 44,45,47,48 37,50
41 Ile4 H Hγ2 1.09 38,39,41,42,43 39,40,41,43 46 44,45,47,48 37
42 Ile4 H y'CH3 0.80 37,38,39,41,42 39,42 47 44,45,46 37,50,51
43 Ile4 H δCH3 0.82 39,41,43 40,41,43 48 45,46 x
44 Ile4 C Cα 58.94 x x 38 39,40,41,42 x
45 Ile4 C Cβ 34.52 x x 39 37,38,40,41,42,43 x
46 Ile4 C Cγ 24.97 x x 40,41 39,42,43 x
47 Ile4 C Cγ' 14.52 x x 42 38,39,40,41 x
48 Ile4 C Cδ 10.73 x x 43 40,41 x
49 Ile4 C CO 171.50 x x x 38,39,50,51,52 x
50 Gly5 H HN 8.79 50,51,52 50,51,52 x 49,53 38,40,41,42,51,52,55
77
Table S4.1 (cont’d)
Residue Nucleus Atom ppm TOCSY COSY HSQC HMBC ROESY
51 Gly5 H Hα1 3.87 50,51,52 50,51,52 53 49,54 42,50,52,55
52 Gly5 H Hα2 3.45 50,51,52 50,51,52 53 49,54 50,51,55
53 Gly5 C Cα 42.17 x x 51,52 50 x
54 Gly5 C CO 168.10 x x x 51,52,55 x
55 Cys6 H HN 8.31 55,56,57,58 55,56 x 54 50,51,52,56,57,58,63
56 Cys6 H Hα 4.94 55,56,57,58 55,56,57 59 60,61 55,58,62
57 Cys6 H Hβ1 3.06 55,56,57,58 56,57,58 60 59,61 16,32,55,58,62
58 Cys6 H Hβ2 2.91 55,56,57,58 56,57,58 60 59,61 55,56,57
59 Cys6 C Cα 49.92 x x 56 57,58 x
60 Cys6 C Cβ 58.80 x x 57,58 56 x
61 Cys6 C CO 167.14 x x x 56,57,58,62 x
62 Asn7 H HN 8.49 62,63,64,65 62,63 x 61,71 16,32,56,57,63
63 Asn7 H Hα 4.77 62,63,64,65 62,63,64,65 68 70 62,64,65,77
64 Asn7 H Hβ1 3.48 63,64,65 63,64,65 69 68,70,71 62,63,65,66
65 Asn7 H Hβ2 3.01 63,64,65 63,64,65 69 68,70 63,64,77
66 Asn7 H δNH2(1) 8.50 66,67 x x 70 64,67
67 Asn7 H δNH2(2) 7.70 66,67 x x 70 15,16,66
68 Asn7 C Cα 50.65 x x 63 64,65 x
69 Asn7 C Cβ 33.05 x x 64,65 x x
70 Asn7 C γCO 173.09 x x x 63,64,65,66,67 x
71 Asn7 C CO 170.01 x x x 62,64 x
72 Pro8 H Hα 4.15 72,73,74,75,76,77,78 72,73,74 79 80,83 1,63,73,76,74
73 Pro8 H Hβ1 2.31 72,73,74,75,76,77,78 72,73,74 80 81,82 72,74,76
74 Pro8 H Hβ2 1.64 72,73,74,75,76,77,78 72,73,74,76 80 79,81,83 1,4,72,73,75
75 Pro8 H Hγ1 1.98 72,73,74,75,76,77,78 x 81 79 x
76 Pro8 H Hγ2 1.81 72,73,74,75,76,77,78 78 81 80,82 72,73,77
77 Pro8 H Hδ1 3.96 72,73,74,75,76,77,78 75,76,77,78 82 79,80,81 63,65,75,76
78 Pro8 H Hδ2 3.60 72,73,74,75,76,77,78 76,77,78 82 81 x
79 Pro8 C Cα 63.15 x x 72 74,75,77 x
80 Pro8 C Cβ 29.65 x x 73,74 72,76,77 x
81 Pro8 C Cγ 24.96 x x 75,76 73,74,77,78 x
82 Pro8 C Cδ 47.30 x x 77,78 73,76 x
83 Pro8 C CO 170.42 x x x 1,72,74 x
78
WORKS CITED
79
WORKS CITED
1. Wieland, T. (1986). Peptides of Poisonous Amanita Mushrooms. Springer: New York.
2. Enjalbert F, Cassanas G, Rapior S, Renault C, and Chaumont JP. (2004). Amatoxins in
wood-rotting Galerina marginata. Mycologia 96(4): 720-729.
3. Sgambelluri RM, Epis S, Sassera D, Luo H, Angelos ER, and Walton JD. (2014). Profiling
of amatoxins and phallotoxins in the genus Lepiota by liquid chromatography combined
with UV absorbance and mass spectrometry. Toxins 6(8): 2336-2347.
4. Smith AH. (1953). New Species of Galerina from North America. Mycologia 45(6): 892-
925.
5. Benedict RG, Tyler VE Jr, Brady LR, and Weber LJ. (1966). Fermentative production of
amanita toxins by a strain of Galerina marginata. J. Bacteriol. 91(3): 1380-1381.
6. Luo H, Hallen-Adams HE, Scott-Craig JS, and Walton JD. (2012). Ribosomal biosynthesis
of α-amanitin in Galerina marginata. Fungal Genet. Biol. 49(2): 123-129.
7. Anderson MR, Nielsen JB, Kitgaard A, Peterson LM, Zacharisa M, Hansen TJ, Blicher LH,
Gotfredsen CH, Larsen TO, Nielsen KF, and Mortensen UH. (2013). Accurate predicion of
secondary metabolite gene clusters in filamentous fungi. Proc. Natl. Acad. Sci. U.S.A.
110(1): E99-107.
8. Brakhage AA and Schroeckh V. (2011). Fungal secondary metabolites - strategies to
activate silent gene clusters. Fungal Genet. Biol. 48(1): 15-22.
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amatoxins and phallotoxins in Amanita phalloides Fr., by high-performance liquid
chromatography. J. Chromatogr. 598(2): 227-236.
10. Kemppainen MJ, and Pardo AG. (2010). Gene knockdown by ihpRNA-triggering in the
ectomycorrhizal basidiomycete fungus Laccaria bicolor. Bioeng. Bugs. 1(5): 354-358.
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Laccaria bicolor using Agrobacterium tumefaciens. Bioeng. Bugs. 1(5): 354-358.
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11(1): 21-32.
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13. Bergman S, Schumann J, Scherlach K, Lange C, Brakhage AA, and Hertweck C. (2007).
Genomics-driven discovery of PKS-NRPS hybrid metabolites from Aspergillus
nidulans. Nat. Chem. Biol. 3(4): 213-217.
14. Luo H, Hong SY, Sgambelluri RM, Angelos E, Li X, and Walton JD. (2014). Peptide
macrocyclization catalyzed by a prolyl oligopeptidase involved in α-amanitin
biosynthesis. Chem. Biol. 21(12): 1610-1617.
15. Lee J, McIntosh J, Hathaway BJ, and Schmidt EW. (2009). Using marine natural products
to discover a protease that catalyzes peptide macrocyclization of diverse substrates. J.
Am. Chem. Soc. 131(6): 2122-2124.
16. May JP and Perrin DM. (2007). Tryptathionine bridges in peptide synthesis. Biopolymers.
88(5): 714-724.
81
CHAPTER 5
BIOCHEMICAL CHARACTERIZATION OF PROLYL
OLIGOPEPTIDASE B AS A PEPTIDE MACROCYCLASE
Note: The content in this chapter has been previously published. Some text has been modified
from the original. Copyright © Elsevier Ltd All rights reserved
Citation: Luo H, Hong SY, Sgambelluri RM, Angelos E, Li X, and Walton JD. (2014). Peptide
macrocyclization catalyzed by a prolyl oligopeptidase involved in α-amanitin biosynthesis.
Chem. Biol. 21(12): 1610-1617.
Author Contributions: Molecular cloning of POPB cDNA was performed by Hong Luo and
Sung-Yong Hong. Evan R. Angelos, Xuan Li, and Hong Luo assisted with POPB purification
and enzyme assays.
82
5.1 Abstract
Amatoxins are ribosomally encoded and post-translationally modified peptides that account for
the majority of fatal mushroom poisonings of humans. A representative amatoxin is the bicyclic
octapeptide α-amanitin, formed via head-to-tail macrocyclization, which is ribosomally
biosynthesized as a 35-amino acid precursor peptide in Amanita spp. and the distantly related
mushroom Galerina marginata. POPB, a member of the prolyl oligopeptidase (POP) family of
serine proteases, has been proposed to play a role in α-amanitin posttranslational processing;
however the exact mechanistic details are not known. Here we show that POPB from G.
marginata is bifunctional and catalyzes two nonprocessive reactions with the α-amanitin
precursor peptide: hydrolysis at an internal Pro residue to release the 10mer N-terminal
sequence, and transpeptidation at a second Pro to produce a cyclic octapeptide composed of the
α-amanitin sequence.
83
5.2 Introduction
A predicted prolyl oligopeptidase (POP) enzyme, GmPOPB, was previously shown by reverse
genetics to be essential for α-amanitin production in the mushroom Galerina marginata (see
Chapter 4). In mushrooms, POPB homologs are only present in species that produce
cycloamanides [1], suggesting that POPB plays a dedicated role in the biosynthesis of
cycloamanides. POPs are large (~80 kDa) serine proteases that hydrolyze peptides at the
carboxyl side of proline residues [2]. POPs have been cloned and characterized from bacteria
[3,4], archaea [5], insects [6], and mammals [7,8,9], and share a conserved two-domain structure
(Figure 5.1). The C-terminal portion of the sequence forms a conserved peptidase domain with
an α/β hydrolase fold and contains the serine protease catalytic triad (Ser-His-Asp) [10,11]. The
N-terminal portion is more variable, but forms a seven bladed β-propeller domain with a
proposed role as a “gating filter” in substrate selection [12,13]. Proline specificity in POPs is
achieved with a hydrophobic S1 specificity pocket and by a ring-stacking interaction with the
indole side-chain of an active site tryptophan residue [12,14].
POP enzymes in bacteria are believed to carry out housekeeping functions in protein turnover
[2]. In the bacterium Kribbella flavida, one POP was shown to be involved in the biosynthesis of
lanthipeptides, RiPPs with antimicrobial activity against Gram-positive bacteria [15]. In
mammals, the majority of known peptide hormones and neuropeptides contain at least one
proline residue [16], and consistent with a role in neuropeptide metabolism, mammalian POP is
concentrated in brain tissue [17]. Aberrant levels of serum POP activity are characteristic of a
number of psychiatric disorders in humans including depression [18], mania, and schizophrenia
[19]. POP inhibitors slow memory loss in Alzheimer’s disease [20] and reverse drug-induced
amnesia in rats [21]. A number of POP-specific inhibitors are in clinical trials [22].
84
Figure 5.1: Crystal Structure of Prolyl Oligopeptidase from Porcine Brain. The
active site is found at the interface of peptidase (purple) and β-propeller (blue) domains.
Reillustrated with PyMol from the Protein Data Bank (http://www.rcsb.org) structure
1H2W.
Cycloamanides are composed of the amino acid sequences from the core domains of their
corresponding precursor peptides [23]. POPB likely functions in proteolysis of the precursors at
the invariable proline residues that separate the core domain from the leader and follower
sequences. Alternatively, POPB could be responsible for backbone macrocyclization of the core
domains, since proteaseses from similar RiPP pathways have recently been shown to catalyze
this reaction [24,25,26,27]. The following studies aim to characterize POPB from G. marginata
in vitro and to define the enzyme’s role in cycloamanide biosynthesis.
85
5.3 Methods
5.3.1 Protein Expression and Purification. GmPOPB cDNA was cloned by Sung Yong Hong
(MSU-DOE Plant Research Laboratory, Michigan State University) and inserted into the pESC-
HIS vector (Agilent Technologies) for expression in Saccharomyces cerevisiae (strain YPH501)
with a N-terminal c-myc epitope tag for purification. Transformed yeast cells were first grown
overnight in SD medium (0.67% yeast nitrogen base without amino acids, 2% dextrose, and
0.13% amino acid mixture minus histidine for selection), and the following morning this culture
was diluted 1:50 with SG media (SD with galactose substituted for dextrose) for induction. Cells
were induced for 48 hr at 30°C with shaking. Cells were harvested at 4,000 x g for 10 min and
then lysed by grinding in liquid nitrogen. The yeast powder was then resuspended in buffer (20
mM Tris, pH 7.5, 0.4% glycerol, 1 mM EDTA, 2 mM DTT) at 100 mL per liter of culture.
Soluble protein was collected at 21,000 x g for 20 min.
Recombinant GmPOPB was first purified on anti c-myc agarose (ThermoFisher) and eluted with
tris-buffered saline (TBS) containing 1 mg/mL c-myc peptide. Ion-exchange was included as a
second purification step on a TSK DEAE-5PW column (Tosoh Bioscience) with a 25 min
gradient from 0 to 600 mM NaCl in 20 mM Tris, pH 7.5 on an Agilent 1100 series HPLC
system. Working enzyme solution was stored in aliquots at -80°C at 1 mg/mL in 20 mM Tris
buffer, pH 7.5, with 2 mM DTT and ~250 mM NaCl. Protein concentrations were measured
using bicinchoninic acid (BCA) (Pierce Biotechnology) against bovine serum albumin (BSA) as
standard. GmPOPB mutants were prepared using a QuikChange Lightning site-directed
mutagenesis kit (Agilent Technologies) and purified in the same manner as wild-type enzyme.
86
5.3.2 Enzyme Assays. POP activity on peptide substrates was assayed in 20 mM Tris HCl (pH
7.5) containing 10 mM dithiothreitol and ~90 µM peptide at 37°C. Chemically synthesized
peptides were supplied by Bachem and Elim Biopharmaceuticals. For kinetic studies, each
reaction contained 15 ng (0.18 pmol) of enzyme and varying amounts of substrate in 50 µL total
volume and triplicate measurements were made for each substrate concentration. Kinetic
constants were calculated using nonlinear curve fitting with GraphPad Prism (GraphPad
Software). At the end of the incubation, methanol was added to 50% (v/v), the samples were
centrifuged at 20,000 x g for 5 min, and the supernatants were dried under vacuum and
resuspended in water. Reactions were analyzed by ESI-LCMS using an Agilent 1100 pump
system and Agilent 6120 single quadrupole mass spectrometer equipped with a multi-wavelength
UV detector. Separation was performed on a reverse-phase C18 column (RS-2546-W185,
Higgins Analytical) with a 20 min linear gradient from 20 mM ammonium acetate (pH 5) to
100% acetonitrile at 1 mL/min. UV absorbance was monitored at 220, 250, and 280 nm.
5.3.3 Product Purification and NMR Spectroscopy. For large-scale purification of the cyclic
reaction product, 5 µg of GmPOPB protein was incubated with 10 mg GmAMA1 peptide
overnight at 37°C. The protein was removed by precipitation with 50% (v/v) methanol and
centrifugation, and the supernatants were dried and resuspended in water. The product was
purified on a preparative C18 column (Supelcosil LC-18, 25 cm x 10 mm, 5 mm) using the same
HPLC method described above at 0.5 mL/min flow rate. Fractions containing product were then
dried to yield ~1.2 mg of white powder (57% yield). LCMS indicated isolation of the intended
product (correct mass and retention time) and ~88% purity on the basis of absorbance at 280 nm.
The purified product was dissolved in DMSO-d6 at 5 mM concentration. NMR spectra were
collected at 25°C on a Varian 600 MHz instrument. 1H atoms were assigned with COSY,
87
TOCSY, and ROESY. TOCSY spectra were acquired with an MLEV17 mixing sequence with a
mixing time (tm) of 80 ms and ROESY spectra were collected with a tm of 200 ms. 13
C atoms
(natural abundance) were assigned with HSQC and HMBC.
88
5.4 Results
5.4.1 Preparation of Recombinant GmPOPB. GmPOPB with an N-terminal c-myc epitope tag
was expressed in S. cerevisiae and purified in two steps from cell extracts. The first step was on
an anti-c-myc agarose affinity column, and the second was by anion exchange on DEAE. The
resulting protein solution gave a single band by SDS-PAGE with the expected molecular weight
(~84 kDa) for the 730 residue protein (Figure 5.2). The method yielded an average of ~1.8 mg
of recombinant protein after the second purification step from one liter of culture. Even after
purification, the protein was highly sensitive to degradation, likely from autoproteolysis, and
required flash freezing and storage in aliquots at -80°C.
Figure 5.2: Purification of Recombinant GmPOPB Expessed in Yeast. Image shows
SDS-PAGE of GmPOPB protein after purification from crude extracts on c-myc agarose
(lane 2) followed by purification by anion exchange chromatography (lane 3).
89
5.4.2 GmPOPB Catalyzes Peptide Macrocyclization. Synthetically produced GmAMA1, the
35mer precursor peptide to α-amanitin and hypothesized natural substrate for GmPOPB, was
incubated with POPB enzyme and the reaction was monitored by LCMS. Activity on the
GmAMA1 peptide was observed (Figure 5.3) with products corresponding to cleavage of the
substrate at proline residues flanking the core domain of AMA1. For the product corresponding
to the core domain sequence, LCMS indicated a monoisotopic mass of 841.4 m/z, 18 fewer mass
units than expected for linearized peptide and suggesting formation of a new peptide bond
concomitant with loss of water and cyclo-IWGIGCNP product.
Figure 5.3: Time Course of Conversion of GmAMA1 to cyclo-IWGIGCNP. Signals
are UV absorbance at 280 nm from HPLC separation of the reaction products between 0
and 90 min of incubation.
90
A large-scale reaction using purified GmPOPB and synthetic GmAMA1 was used to produce
~1.2 mg of the putative cyclic reaction product for NMR experiments. 1H and
13C atoms in the
product were assigned with COSY, TOCSY, ROESY, HSQC, and HMBC experiments (Figures
S5.2 through S5.6). A signal was observed in the HMBC experiment corresponding to through-
bond coupling between the backbone amide of Ile1 and carbonyl of Pro8, and served as direct
detection of the newly formed peptide bond in the product (Figure 5.4). Coupling between the
HN of Ile1 and Hα of Pro8 was also observed in the ROESY experiment, consistent with their
close proximity upon cyclization. Finally, the free thiol proton from Cys6 was able to be
assigned, indicating that the product did not contain an internal thioester, a modification that
could result in the same 18 unit mass discrepancy. These studies confirm the formation of cyclo-
IWGIGCNP and a macrocyclization reaction catalyzed GmPOPB.
Figure 5.4: Amide Bond Couplings in the HMBC Spectrum of cyclo-IWGIGCNP. In
the HN-CO region of the spectrum, a signal (highlighted in red) indicating through-bond
coupling between Ile1 and Pro8 residues confirms a cyclized backbone in the reaction
product.
91
5.4.3 GmPOPB is a Bifunctional Enzyme. POPB was incubated with excess AMA1 and
analyzed before the reaction was complete and the substrate was consumed. AMA1 was
converted to a series of products consistent with bifunctional hydrolase and macrocyclase
activity by POPB (Figure 5.5). Specifically, the data indicate hydrolysis at the first proline
residue (Pro10) and transpeptidation/cyclization at the second (Pro18) in the AMA1 sequence. A
small amount of linearized IWGIGCNP peptide was also detectable as product, but less than the
limit of detection for UV absorbance and therefore less than 1% of total product composed of the
core domain sequence. During the reaction, a truncated 25mer intermediate resulting from
hydrolysis at Pro10 and removal of the N-terminal leader sequence accumulated transiently
(Figure 5.5). Since no product corresponding to initial activity at Pro18 was observed, the two
reaction steps catalyzed by POPB are ordered, with hydrolysis preceding cyclization (Figure
5.6). To determine if cyclization requires concurrent hydrolysis or if the steps are exclusive,
GmPOPB was incubated separately with the 25mer intermediate as initial substrate. As with full-
length substrate, cyclo-IWGIGCNP was produced, indicating the reaction steps are non-
processive (Figure S5.6).
The kinetic constants Km, Vmax, and kcat were determined for both full-length and truncated
substrates by measuring rates of cyclic product formation at varying substrate concentrations
(Figure 5.7 and Table 5.1). Cyclo-IWGIGCNP was produced from both substrates at identical
rates of 5.7 sec-1
consistent with cyclization being rate-limiting in the overall reactionand
supported by the observed build-up of the 25mer intermediate in time-course assays. Backbone
macrocyclization of peptides is catalyzed by three other known enzymes that have been
biochemically characterized: PatG, PCY1, and butelase, with turnover rates of 1 hr-1
, 2 hr-1
, and
17 sec-1
respectively. POPB is comparable in efficiency to butelase.
92
Figure 5.5: Reaction Products from GmPOPB Activity on GmAMA1. Products were
analyzed by LCMS before the reaction went to completion. The observed products and
intermediate formed indicate an ordered, two-step reaction scheme beginning with N-
terminal hydrolysis. Signals are UV absorbance at 280 nm (top) and extracted ion
chromatograms (bottom) for the expected monoisotopic masses of substrate and each
product. The observed m/z values and charges are indicated.
Figure 5.6: Two-step Nonprocessive Reaction Catalyzed by POPB on the α-
Amanitin Precursor Peptide.
93
Figure 5.7: Kinetic Analysis of GmPOPB. Shown are overlaid GmPOPB saturation
curves with 35mer (blue) and 25mer (red) substrates.
Table 5.1: GmPOPB Cyclization Kinetic Constants with 35mer and 25mer
GmAMA1 Substrates.
94
5.4.4 Residues Involved in Macrocyclization. GmPOPB is 75.5% identical to AbPOPB, the
POPB homolog from Amanita bisporigera; 59% identical to GmPOPA, a housekeeping POP
uninvolved in cycloamanide biosynthesis [1]; and 37% identical to the two well characterized
POP enzymes from porcine brain and muscle tissue. To identify residues or motifs that may be
involved in POPB’s capacity for macrocyclization, the sequences of the porcine POPs and
POPAs from A. bisporigera and G. marginata were analyzed in a ClustalW2 multiple sequence
alignment (Figure S5.7). A high degree of similarity between all six POPs is revealed in the
alignment. All six proteins are roughly 750 amino acids in length with no apparent gaps or
additional motifs present that are unique to POPB. Consistent with a serine protease mechanism,
all the fungal POPs contained serine, aspartic acid, and histidine residues (Ser577, Asp661, and
His698 in GmPOPB) that aligned with these same catalytic residues in the porcine POPs [12].
The active site Trp residue shown in crystallization studies with the porcine POPs to stack with
proline in the substrate also aligned with Trp residues (Trp619 in GmPOPB).
A GmPOPB variant (S577A) lacking the predicted catalytic serine was prepared to test whether
the residue is required for initial hydrolysis of AMA1 and also to test its involvement in
cyclization. No activity was observed with the GmPOPB(S577A) variant on either the full-length
35mer AMA1 peptide or the 25mer intermediate missing the leader sequence. This supports the
classification of POPB as a serine protease and the involvement of Ser577 in the N-C cyclization
mechanism. The sequences adjacent to the catalytic Asp and His also contain residues that are
differentially conserved between the POPBs and the other POPs (Table 5.2), and these residues
are hypothesized to play a role in cyclization.
95
Table 5.2: Differentially Conserved Residues Between POPA and POPB. Sequences
in POPA (red) and POPB (green) are located adjacent to catalytic Asp and His residues
(purple)
Region 1 (Asp661) Region 2 (His698)
POPA ADHDDRVVP -KAGHGMGK
POPB NIGDGRVVP SWLGHGMGK
96
5.5 Discussion
The enzyme POPB was determined to function in both leader peptide removal and head-to-tail
macrocyclization during the biosynthesis of α-amanitin and likely all other cycloamanides. The
enzyme catalyzed the two-step, non-processive reaction shown with the precursor peptide to α-
amanitin as substrate. A total of four other enzymes, all predicted serine or asparagine proteases
involved in similar RiPP pathways, have been shown to catalyze peptide head-to-tail
condensation/macrocyclization: PatG [24], PCY1 [25], butelase [26], and AEP1 [27]. POPB is
unique among these enzymes in its bifunctionality. While the other cyclases require the leader
sequence to first be removed from the precursor peptide substrate by a separate protease, POPB
catalyzes both steps. The catalytic Ser577 residue in POPB was found to be necessary for both
hydrolase and cyclase activities, and catalysis is therefore hypothesized to involve a familiar
serine protease mechanism in which macrocyclization is achieved through removal of the
covalent intermediate via deacylation with the N-terminal amine of bound substrate instead of
water (Figure 5.8). Further mutagenesis and structural studies with bound substrates will be
necessary for a complete description of the mechanisms utilized by this unusual enzyme.
Figure 5.8: Hypothetical Mechanism for Macrocylization Catalyzed by POPB.
97
APPENDIX
98
APPENDIX
Figure S5.1: 2D gCOSY Spectrum of cyclo-IWGIGCNP.
Figure S5.2: 2D TOCSY Spectrum of cyclo-IWGIGCNP.
99
Figure S5.3: Amide Region from TOCSY Spectrum of cyclo-IWGIGCNP.
The 1H assignments are indicated.
100
Figure S5.4: 2D ROESY Spectrum of cyclo-IWGIGCNP.
Figure S5.5: 2D 1H-
13C HSQC Spectrum of cyclo-IWGIGCNP.
101
Figure S5.6: Formation of cyclo-IWGIGCNP Product from 35mer and 25mer Substrates.
Signals are extracted ion chromatograms (EICs) for the expected masses of the peptides. The
observed m/z values and charge states (z) are indicated.
102
Figure S5.7: Multiple Sequence Alignment of POPB and Other Prolyl Oligopeptidases.
Positions with conserved/identical residues (orange) and non-identical but conserved by amino
acid properties (yellow) are highlighted. Residues that are differentially conserved between
POPB (green) and homologs without cyclase activity (red) are also indicated, as well as the
catalytic Ser-His-Asp triad and tryptophan residue critical for proline specificity (purple).
103
Figure S5.7 (cont’d)
104
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CHAPTER 6
VERSATILITY OF PROLYL OLIGOPEPTIDASE B IN
PEPTIDE MACROCYCLIZATION
Note: The content in this chapter has been previously published. Some text has been modified
from the original.
Citation: Sgambelluri RM, Smith MO, and Walton JD. (in press). Versatility of prolyl
oligopeptidase B in peptide macrocyclization. ACS Syn. Biol. doi: 10.1021/acssynbio/7b00264
Author Contributions: Preparation of DNA constructs for expression of POPB substrates was
performed by Miranda O. Smith.
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6.1 Abstract
Cyclic peptides are promising compounds for new chemical biological tools and therapeutics due
to their structural diversity, resistance to proteases, and membrane permeability. Amatoxins, the
toxic principles of poisonous mushrooms, are biosynthesized on ribosomes as 35mer precursor
peptides, which are ultimately converted to hydroxylated bicyclic octapeptides. The initial
cyclization steps, catalyzed by a dedicated prolyl oligopeptidase (POPB), involves removal of
the 10-amino acid leader sequence from the percursor peptide and transpeptidation to produce a
monocyclic octapeptide intermediate. The utility of POPB as a general catalyst for peptide
cyclization was systematically characterized using a range of precursor peptide substrates
produced either in E. coli or chemically. Substrates produced in E. coli were expressed either
individually or in mixtures produced by codon mutagenesis. A total of 127 novel peptide
substrates were tested, of which POPB could cyclize 100. Peptides of 7-16 residues were
cyclized at least partially. Synthetic 25mer precursor peptide substrates containing modified
amino acids including D-Ala, β-Ala, N-methyl-Ala, and 4-hydroxy-Pro were also successfully
cyclized. Although a phalloidin heptapeptide with all L amino acids was not cyclized, partial
cyclization was seen when L-Thr at position #5 was replaced with the naturally occurring D
amino acid. POPB should have broad applicability as a general catalyst for macrocyclization of
peptides containing 7 to at least 16 amino acids, with an optimum of 8-9 residues.
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6.2 Introduction
Due to their structural ridigity and conformational diversities, cyclic peptides often display high
affinity binding to target macromolecules, relatively high membrane permeability, and resistance
to proteases [1-4]. Nine cyclic peptide drugs have been approved in the past ten years against
bacterial and fungal infections, cancer, and gastrointestinal disorders [5]. Recent examples of
promising cyclic peptide drug leads include an inhibitor of the RAS oncogene [6]; the modified
griselimycins, which have promise against multidrug resistant tuberculosis [7]; a cyclotide that
activates the p53 tumor suppressor pathway [8]; and lugdunin, a novel antibiotic from a human
commensal bacterium that is active against Staphylococcus aureus [9]. However, synthesis of
cyclic peptides remains difficult and expensive compared to linear peptides [10].
Ribosomally biosynthesized cyclic peptides, known as RiPPs, have been described from bacteria,
plants, mammals, and fungi [11]. Prior to the discovery of the genes encoding the amatoxins,
phallotoxins, and other cyclic peptides from the agaric genus Amanita (collectively known as the
cycloamanides), RiPPs were unknown in fungi [12-14]. Amatoxins such as α-amanitin are
defining inhibitors of RNA polymerase II, and phallotoxins such as phalloidin bind and stabilize
F-actin [15-17]. The amatoxins are highly stable and rapidly absorbed into the bloodstream and
into mammalian cells [18].
Cycloamanides are biosynthesized initially as small (33-37 amino acid) precursor peptides
encoded by a gene family comprising at least 73 members among different Amanita species
[12,19,20]. The conserved structures of the cycloamanide precursor peptides are composed of a
10-amino acid leader, a variable region of 6-10 amino acids which give rise to the mature toxins,
and a conserved follower peptide of 17 residues. Although the amino acid content of the variable
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region in the naturally occurring cycloamanide gene family is biased toward hydrophobic amino
acids and especially Pro, all 20 amino acids are present in at least one predicted cycloamanide
[20].
Cyclization of the variable region of the cycloamanides occurs in two nonprocessive steps, both
catalyzed by a specialized prolyl oligopeptidase, POPB [21]. The amatoxins and phallotoxins,
but not the classic monocyclic cycloamanides, are further posttranslationally processed by
multiple hydroxylations and formation of a cross-bridge between Cys and Trp called
tryptathionine [22]. Additional modifications include sulfoxidation in the amatoxins and
epimerization of one amino acid in the phallotoxins [18].
The kinetic efficiency of POPB from Galerina marginata expressed in Saccharomyces
cerevisiae is sufficiently high to make it a practical reagent for custom synthesis of cyclic
peptides [21]. POPB is comparable in catalytic properties to the peptide macrocyclase butelase 1
from Clitoria ternatea and PCY1 from Saponaria vaccaria [23-26]. Detailed kinetic studies on
POPB expressed in E. coli confirmed its high catalytic efficiency as a peptide macrocyclase and
showed that release of the follower peptide is the limiting step [23]. Here we explore the utility
of POPB as a general catalyst for peptide macrocyclization through characterization of the
enzyme’s substrate versatility and limitations on composition and length of the core domain
sequence.
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6.3 Methods
6.3.1 DNA Constructs. A cDNA of the AMA1 precursor peptide gene from G. marginata
(GmAMA1) was synthesized from total RNA and cloned into the pMAL-c5x expression vector
(containing the gene for maltose binding protein and a Factor Xa protease cleavage site) (New
England Biolabs) using the In-Fusion HD cloning system (Clontech). Constructs for expression
of AMA1 variants with single amino acid substitutions were prepared by site-directed
mutagenesis of the wild-type construct using a QuikChange Lightning kit (Agilent
Technologies). Coding sequences for natural substrates and substrates with varying core domain
lengths were obtained as synthetic gene fragments (gBlocks, Integrated DNA Technologies) and
inserted into the expression vector using In-Fusion. All DNA constructs were verified by Sanger
sequencing and transformed into E. coli (BL21-DE3) cells for expression.
6.3.2 Preparation of POPB Substrates. Substrates containing unusual amino acids were
produced by solid-phase synthesis by Bachem Americas, Inc. For all other peptide substrates, E.
coli cells expressing MBP-peptides were grown with ampicillin selection in Luria broth (LB)
supplemented with 2 g/L glucose at 37˚C with shaking and induced at an OD600 of approximately
0.6 with 2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 4 hr at 30˚C. The cultures
were harvested by centrifugation at 8,000 x g for 10 min and the pellet was resuspended in buffer
(20 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA) and flash frozen. Cells were lysed by thawing
at 42˚C in the presence of 1 mg/mL lysozyme and 0.5 mM PMSF. DNase (10 units/mL) was
added until the viscosity of the solutions cleared, and insoluble material was removed by
centrifugation at 21,000 x g for 20 min. The MBP-peptide fusions were isolated from crude
extracts on amylose resin (New England Biolabs) and eluted with 10 mM maltose. Eluates were
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then concentrated to 100-fold their original volume with Macrosep Advance spin concentrators
(Pall Life Sciences) and incubated at room temperature with 8 μg/mL Factor Xa protease (New
England Biolabs). After protease treatment, MBP was precipitated with 50% (v/v) methanol and
removed by centrifugation, and the peptide solutions were dried under vacuum and redissolved
in water containing 2 mM DTT.
6.3.3 Cyclization Assays and LCMS. Cyclization assays were typically performed in 20 mM
Tris, pH 7.5 with 25 mM DTT, ~25 μg substrate, and 5 μg enzyme (prepared as described in
Section 5.3.1) at 37˚C. Wild-type AMA1 peptide was used as a control. After 4 hr, the enzyme
was removed by precipitation with methanol and the reactions were dried under vacuum and
redissolved in water. The products were analyzed by LCMS using an Agilent 1200 pump system
and an Agilent 6120 single quadrupole instrument in positive ion mode with a 20 min gradient
from 20 mM NH4OAc (pH 5) to acetonitrile on a Higgins Proto-300 C18 column. For each
substrate, reactions were analyzed before and after addition of POPB by UV/Vis and extracted
ion chromatograms (EICs) targeting the expected masses of full-length substrate, the expected
cyclic peptide product, and the expected linear form of the core domain. Substrate levels and the
relative amounts of cyclic vs. linear product were quantitated by integrating peak areas at OD280
with a detection limit of 0.15 μmol/L per tryptophan residue. Relative concentrations of Trp-
noncontaining peptides were estimated from absorbance at 220 nm.
6.3.4 Library Preparation and Analysis. The plasmid contruct for library production was
prepared with an Ultramer ssDNA fragment (Integrated DNA Technologies) that contained a
sequence encoding full-length precursor peptide with degenerate codons for the core domain
sequence, i.e., XW(G/A)X(G/A)CXP, as well as forward and reverse adaptor sequences for
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downstream cloning. Complementary strands for the ssDNA mixture were synthesized in a
primer extension reaction using T4 polymerase and the resulting products were inserted into the
pMAL vector by In-Fusion. BL21(DE3) cells were transformed with the resulting plasmids and
120 colonies were selected for Sanger sequencing. Colonies giving viable sequences were
collected and separated into ten groups based on expected product masses, with no duplicate
masses present within the same group.
Colonies were grown separately overnight and then pooled for growth and induction in 50 mL
cultures of LB. For each polyculture, the remaining processing steps from growth to cyclization
were identical to those used for preparation of individual precursor peptide substrates. The ten
product mixtures were analyzed using LCMS, with EICs for the expected substrate and product
(both linear and cyclic) masses. Native AMA1 was included in all experiments as a standard for
cyclization efficiency and background. All products concluded to be present within the mixtures
gave EIC signals not observed in the background nor in the absence of POPB treatment and
corresponded to monoisotopic masses of the correct charge state.
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6.4 Results
6.4.1 Enzyme and Substrate Preparation. Recombinant POPB enzyme from G. marginata was
produced in yeast with an N-terminal myc epitope tag and purified on anti-c-myc agarose
followed by anion exchange chromtography, as described previously [21]. As a source of
precursor peptide substrates, a strategy was developed for their expression in E. coli. The coding
sequence for the amanitin precursor peptide (AMA1) from G. marginata was expressed as a
maltose-binding protein (MBP) fusion by cloning into the vector pMAL-c5x. This afforded high
stability, yields, and tractability of the precursor peptides. After induction of expression, the
MBP fusion proteins were purified from cell extracts on amylose resin. Treatment with Factor
Xa protease released the GmAMA1 peptide from the C-terminus of MBP, and the MBP was then
removed by precipitation in methanol. LCMS indicated the release of GmAMA1 from the fusion
protein upon treatment with Factor Xa and formation of cyclo-IWGIGCNP upon addition of
GmPOPB enzyme. Approximately 6 mg of precursor peptide was produced from one liter of
bacterial culture (Figure 6.1).
6.4.2 Amino Acid Preferences for Cyclization. Site-directed mutagenesis of the wild-type
AMA1 expression construct was used to generate a series of mutants with amino acid
substitutions at each position of the core domain (sequence IWGIGCNP), excluding Pro8, which
was presumed to be essential for POPB recognition. Reactions contained 5 μg POPB and 25 μg
substrate, and ran for 4 hr at 37˚C. The results are summarized in Table 6.1 and the
corresponding LCMS chromatograms are shown in Figures S6.1-S6.7. Cyclic products were
produced from all 28 substrates. All substitutions to residues #1, #4, #6, and #7 gave yields of
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>99% of cyclized core domain. Some substitutions at positions #2, #3, and #5 were less tolerated
and gave higher yields of linear octamer product resulting from preference of
Figure 6.1: Expression and Purification of the Amanitin Precursor Peptide. (A)
SDS-PAGE of AMA1 precursor peptide expression and purification as a MBP fusion.
(B) LCMS indicating relase of AMA1 peptide by Factor Xa protease and formation of
cyclo-IWGIGCNP by POPB.
the enzyme for hydrolysis over transpeptidation in the second catalytic step. Decreased yields
were observed when residue #2 was changed to polar amino acids Ser or Asn, suggesting a
preference for nonpolar residues at this position. POPB tolerated Ala but not Ser, Leu, or Asn at
positions #3 and #5. The cyclic product yields for these less preferred substrates ranged from
18% (G3L) to 76% (G3S).
While the incubation time used in these assays was intended to allow the reactions to run to
completion, detectable amounts of full-length substrate remained in the assays with five of the
mutants (G3S, G3L, G3N, G5S, G5L), all of which also gave reduced yields of cyclic product.
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Position AA Type Residue Rxn Progress (%) Cyclic (%)
1 wild type Ile > 99 > 99
1 small, nonpolar Ala > 99 > 99
1 small, polar Ser > 99 > 99
1 large, nonpolar Leu > 99 > 99
1 large, polar Asn > 99 > 99
2 wild type Trp > 99 > 99
2 small, nonpolar Ala > 99 > 99
2 small, polar Ser > 99 32
2 large, nonpolar Phe > 99 > 99
2 large, polar Asn > 99 46
3 wild type Gly > 99 > 99
3 small, nonpolar Ala > 99 > 99
3 small, polar Ser 72 76
3 large, nonpolar Leu 33 18
3 large, polar Asn 85 63
4 wild type Ile > 99 > 99
4 small, nonpolar Ala > 99 > 99
4 small, polar Ser > 99 > 99
4 large, nonpolar Leu > 99 > 99
4 large, polar Asn > 99 > 99
5 wild type Gly > 99 > 99
5 small, nonpolar Ala > 99 > 99
5 small, polar Ser 88 74
5 large, nonpolar Leu 92 60
5 large, polar Asn > 99 64
6 wild type Cys > 99 > 99
6 small, nonpolar Ala > 99 > 99
6 small, polar Ser > 99 > 99
6 large, nonpolar Leu > 99 98
6 large, polar Asn > 99 97
7 wild type Asn > 99 > 99
7 small, nonpolar Ala > 99 > 99
7 small, polar Ser > 99 > 99
7 large, nonpolar Leu > 99 > 99
7 large, polar Gln > 99 > 99
Table 6.1: Tolerance of POPB for Amino Acid Substituions in the Core Region of
AMA1. Wild-type sequnces are coded green, reactions that gave reduced yields of cyclic
product are coded pink. Corresponding LCMS traces are shown in Figures S6.1-S6.7.
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To test whether the reduced cyclization efficiency was due to reduced first-stage hydrolysis,
25mer forms (i.e., without the 10-amino acid leader) of four of the sequences (wild-type, W2S,
G3L, and G5S) were tested as substrates. POPB cyclase is as efficient with the 25mer as with the
native 35mer [21]. The same efficiencies in cyclization were observed with the 25mer substrates
(Table S6.1). Thus, these substitutions resulted in poorer substrates for both hydrolysis and
cyclization steps.
6.4.3 Cyclization of Sequences Containing Unusual Amino Acids. Amatoxins and
phallotoxins contain up to five hydroxylations. Both groups of toxins have 4-hydroxyproline,
which is critical for high affinity binding of α-amanitin to pol II [17]. The amatoxins also contain
6-hydroxytryptophan, which is the preferred site for attachment of antibodies in antibody-
amanitin conjugates targeted against cancer cells [27]. It is not known whether the
hydroxylations occur before or after cyclization by POPB. In either case, cyclizing the amanitin
percursor with the Pro and Trp hydroxylations already in place would facilitate great progress
towards the complete in vitro biosynthesis of α-amanitin, which to date has eluded chemical
synthesis. Furthermore, the compatability of POPB with unusual amino acids such as N-
methylated amino acids and/or β-amino acids would expand the utility of POPB to make novel
cyclic peptides.
We chemically synthesized four additional substrates that contained the modified amino acids
trans-4-hydroxyproline, 5-hydroxytryptophan, N-methylalanine, and β-alanine (an Fmoc
derivative of 6-hydroxytryptophan was not commercially available). These substrates were
prepared as the 25mer form lacking the N-terminal leader domain. All four of these substrates
were cyclized by POPB (Figure 6.2). Reduced yields were observed from the substrate
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containing N-methylalanine at position #3 of the core domain, which gave primarily linearized
product. After 4 hr, 26% of the substrate containing 4-hydroxyproline remained, indicating that
both hydrolysis and transpeptidation of this substrate was less efficient. POP enzymes achieve
proline specificity through a ring stacking interaction between Pro and an active site Trp [28],
and this interaction might have been adversely affected by the hydroxyl group. The results
indicate that POPB can tolerate amino acids beyond the proteinogenic twenty.
Figure 6.2: Cyclization of Peptides Containing Unusual Amino Acids. The modified
residues are highlighted in red. Synthetic linear 25mers were incubated with POPB and
the reactions analyzed by LCMS. Shown are overlaid EICs; substrate (S) signals are
shown in green, cyclized core domains (C) in red, and linearized core domains (L) in
blue. Values in the table are the amount of product present as cyclized core domain as a
percentage of total cyclic + linear products.
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6.4.4 Core Domain Length Requirement. Naturally occurring cycloamanides in Amanita
species contain 6 to 10 amino acids [18,20]. To examine the allowed peptide lengths for
cyclization by POPB in vitro, we prepared six precursor peptides in E. coli with core domains
ranging from 6 to 16 residues. Core domains with less than 8 residues were prepared by
removing amino acids from the wild-type AMA1 sequence. For longer sequences, Gly, Ala, and
Val were added due to their small size and passive nature, and Ser was included in the 16mer
sequence to avoid possible issues with water insolubility. Cyclization occurred for all tested
substrates with longer core domains (9mer, 10mer, 12mer, and 16mer) (Figure 6.3). Longer
sequences were less efficiently cyclized, but even the 16mer yielded 42% cyclic product with
some unreacted substrate. Hexamer and heptamer core peptides were efficiently processed but
only linear products were produced.
6.4.5 Synthesis of Naturally Occurring Cycloamanides. Amanita phalloides and A.
bisporigera produce a number of homodetic monocyclic hexa- to decapeptides, of which six
have been structurally characterized [18,20]. The known mushroom genomes predict that these
fungi produce more than 50 additional cycloamanides [19,20]. We tested cyclization of POPB
substrates containing the sequences of several cycloamanides produced by expression in E. coli,
as well as sequences for the precursors of β-amanitin (i.e., α-amanitin in which Asp7 replaces
Asn7), and two phallotoxins, phallacidin (PHA; core sequence AWLVDCP) and phalloidin
(PHD; core sequence AWLATCP).
As before, only linearized products from sequences shorter than eight residues were observed
(i.e., CyalA, CylB, PHA, and PHD) (Table 6.2, Figure S6.8). The N-terminal leader peptide of
the substrate containing the phallacidin (PHA) sequence was hydrolyzed to
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Figure 6.3: POPB Products Produced from Substrates with Varying Core Domain
Lengths. “% Cyclic” is the amount of product produced as cyclized core domain as a
percentage of total cyclic + linear product. Shown are overlaid extracted ion
chromatograms (EICs) for substrate (S) in green, cyclized core domains (C) in red, and
linearized core domains (L) in blue.
yield the 25mer, but no further hydrolase or cyclase activity was observed. The inability of
POPB to cyclize these shorter sequences was unexpected, since Amanita mushrooms make
cyclic hexapeptides (CylA) and heptapeptides (CylB and phallotoxins). Possible explanations are
that other steps such as hydroxylation or epimerization occur before, and are required for,
cyclization by POPB, or that the enzyme from Galerina has more limited substrate versatility
than POPB from Amanita species.
All naturally occurring sequences with at least eight residues were cyclized with good yields
including the β-amanitin sequence (Table 6.2, Figure S6.8). The decamer antamanide sequence
122
was cyclized slowly, with less than 10% of the substrate being consumed after 4 hour incubation.
Overall, these results show that POPB could be useful to produce at least some of the natural
cycloamanides, which have immunosuppressant and other biological activities but are currently
only available in limited quantities from mushroom extracts [29,30]. The CylD sequence
(MLVFLPLP) gave cyclic and no linear product despite the presence of a bulky Leu residue at
position #5, which caused reduced yields in the assays with AMA1 single mutants (Table 6.1).
This result indicates that amino acid preferences for cyclization cannot be defined strictly by
position in the substrate, but are instead influenced by overall sequence. For instance, mutations
to the Gly residues in the α-amanitin sequence might have led to reduced yields due to a loss of
flexibility in the sequence, while the turn-inducing effect of the internal Pro in the CylD
sequence might facilitate cyclization.
Table 6.2: Cyclization of Naturally Occurring Cycloamanides. Corresponding LCMS
traces are shown in Figure S6.8.
Footnotes: aSubstrate containing the PHA sequence was hydrolyzed to the 25mer form
but no futher processing occurred. b24 hour incubation.
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6.4.6 Cyclization of the Phalloidin Sequence with D-threonine. All of the natural phallotoxins
contain one D amino acid at position #5, either D-Asp in phallacidin or D-Thr in phalloidin [18].
Introduction of D-amino acids into peptide sequences can improve the efficiency of cyclization
[31]. Since no cyclization was observed with the phallotoxin precursor substrates containing all
L amino acids (i.e., PHA and PHD), we hypothesized that epimerization might occur
biosynthetically prior to cyclization and therefore promote cyclization. A substrate containing the
phalloidin sequence (AWLATCP) with D-Thr was produced synthetically. The presence of D-
Thr resulted in formation of a significant level (13%) of the corresponding cyclic product
whereas the all L version showed only hydrolysis of the substrate to the linear octapeptide and no
cyclization (Figure 6.4). This demonstrates that POPB can cyclize peptides smaller than eight
residues, albeit at low efficiency under our standard conditions, and suggests that epimerization
in the phallotoxins might occur prior to cyclization, i.e., at the precursor peptide stage or after
removal of the leader peptide.
6.4.7 Cyclic Peptide Library Production. As a more rapid strategy for assessing the substrate
versatility of POPB, we constructed a model library of cyclic peptides and processed them in
batches. A trial experiment was first performed using ten of the previously prepared AMA1
substrates with single substitutions that gave products with different masses and retention times.
Inoculating the growth medium with ten individual colonies directly from agar plates resulted in
inconsistent formation of the expected products, likely due to unequal growth rates among the E.
coli strains or differences in inoculation sizes. Consistent production of all ten products could be
obtained by first growing the cultures separately before pooling for induction. After induction,
the pooled cultures were processed through POPB cyclization en masse. The overall scheme is
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Figure 6.4: LCMS Comparing POPB Products Produced from Substrates
Containing the PHD Sequence with either L-Thr or D-Thr. Shown are overlaid
extracted ion chromatograms (EICs); linear products (L) are shown in blue, cyclic
products (C) in red, and the observed m/z values are indicated.
illustrated in Figure 6.5. The results showing expression of all ten cyclic peptides produced in a
single batch are shown in Figure S6.9.
For a randomized library, DNA molecules with degenerate core domains and conserved leader
and follower domains were synthesized as single-stranded DNA with mixed nucleotides in the
core sequence to encode X-W-(G/A)-X-(G/A)-C-X-P, where X is any amino acid. X was
encoded by NNK, where N is any nucleotide, and K is guanine or thymidine. This allows
encoding of all possible amino acids but eliminates two of the three stop codons. Positions #3
and #5 were encoded as either Gly or Ala (codon G[C/G]A) to maximize cyclization efficiency,
and the Trp2 and Cys6 residues were maintained to permit the future possibility of tryptathionine
125
formation. Complementary strands for the ssDNA template mixture were synthesized in a primer
extension reaction, the products were inserted into the same pMAL expression vector used for
expression of the individual substrates, and E. coli cells were transformed with the resulting
plasmids. Transformants were randomly selected and their plasmid inserts sequenced. Of 120
inserts sequenced, 79 (66%) gave viable sequences encoding potential POPB substrates. The 41
nonviable sequences contained frameshifts, deletions, stop codons, or sequence errors likely
introduced during complementary strand synthesis.
Figure 6.5: Scheme for Generating Mixed Cyclic Peptide Libraries.
126
The colonies expressing viable substrates were grown separately in overnight starter cultures and
then pooled into polycultures for expression in ten separate groups, chosen so that no two cyclic
products of the same mass would be present within each final mixture. The remaining
preparation steps of protein extraction, column purification, isolation of the 35mer precursor
peptide substrates from MBP-fusions, and in vitro cyclization with POPB were then carried out
en masse as outlined in Figure 6.5.
The resulting peptide mixtures were analyzed by LCMS and extracted ion analysis for the
predicted masses of the cyclic peptides. Cyclic products from 58 of the 79 substrates were
confirmed within the mixtures (Figures S6.10). All 20 proteinogenic amino acids were
represented among the product sequences. The 21 cyclic products that were expected but absent
from the mixtures fell into two categories: either they contained charged residues (17 total) or
they contained Tyr at the first position (4 total). However, other substrates with these same
characteristics were successfully cyclized and therefore no firm rules for POPB substrate
requirements could be established. No full-length substrate, 25mer intermediate, or linear
product were detected for many of the charged compounds and for none of the peptides
containing Tyr. The absence of these compounds in the final cyclized pool might be due to
problems during E. coli expression or purification of the precursor peptides and not POPB
cyclization.
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6.5 Discussion
Tables S6.2 and S6.3 include a list of the core peptide sequences tested (127 total) and the cyclic
peptides successfully produced (100 total) in this study. The pilot library study demonstrated the
feasibility of producing cyclic peptides in batches, which in principle could be scaled up to at
least hundreds. Additional time could be saved by not prescreening the plasmid inserts by DNA
sequencing, or by not growing the strains separately before induction.
POPB is a versatile and efficient peptide macrocyclase that could be used to make billions of
novel cyclic peptides of 8-16 amino acids including unusual amino acids. Amanitin has recently
been shown to be a promising “warhead” in antibody-drug conjugates against colorectal and
prostate cancers [27,32], but currently the only source of amanitin is from mushrooms collected
in the wild. Our demonstration that key hydroxylations (on Pro and Trp) that occur in native
amatoxins and phallotoxins can be preintroduced into the substrates of POPB might also
facilitate the development of a synthetic or semisynthetic approach to α-amanitin production.
128
APPENDIX
129
APPENDIX
Table S6.1: Compared Cyclization Yields of 35mer and 25mer Substrates. Values are
percentage of total core domain present as cyclic peptide in the final products.
Figure S6.1: Effect of Single Amino Acid Substitions on Cyclization by POPB at Position 1
of the AMA1 Core Domain. Each trace shows overlaid EICs before (top) or after (bottom)
POPB treatment. Substrate (S) signals are shown in green, cyclized core domains (C) in red, and
linearized core domains (L) in blue.
wild-type W2S G3L G5S
35mer > 99 32 ± 3 18 ± 5 74 ± 4
25mer > 99 30 ± 4 19 ± 3 77 ± 5
130
Figure S6.2: Effect of Single Amino Acid Substitions on Cyclization by POPB at Position 2
of the AMA1 Core Domain. Each trace shows overlaid EICs before (top) or after (bottom)
POPB treatment. Substrate (S) signals are shown in green, cyclized core domains (C) in red, and
linearized core domains (L) in blue.
131
Figure S6.3: Effect of Single Amino Acid Substitions on Cyclization by POPB at Position 3
of the AMA1 Core Domain. Each trace shows overlaid EICs before (top) or after (bottom)
POPB treatment. Substrate (S) signals are shown in green, cyclized core domains (C) in red, and
linearized core domains (L) in blue.
132
Figure S6.4: Effect of Single Amino Acid Substitions on Cyclization by POPB at Position 4
of the AMA1 Core Domain. Each trace shows overlaid EICs before (top) or after (bottom)
POPB treatment. Substrate (S) signals are shown in green, cyclized core domains (C) in red, and
linearized core domains (L) in blue.
133
Figure S6.5: Effect of Single Amino Acid Substitions on Cyclization by POPB at Position 5
of the AMA1 Core Domain. Each trace shows overlaid EICs before (top) or after (bottom)
POPB treatment. Substrate (S) signals are shown in green, cyclized core domains (C) in red, and
linearized core domains (L) in blue.
134
Figure S6.6: Effect of Single Amino Acid Substitions on Cyclization by POPB at Position 6
of the AMA1 Core Domain. Each trace shows overlaid EICs before (top) or after (bottom)
POPB treatment. Substrate (S) signals are shown in green, cyclized core domains (C) in red, and
linearized core domains (L) in blue.
135
Figure S6.7: Effect of Single Amino Acid Substitions on Cyclization by POPB at Position 7
of the AMA1 Core Domain. Each trace shows overlaid EICs before (top) or after (bottom)
POPB treatment. Substrate (S) signals are shown in green, cyclized core domains (C) in red, and
linearized core domains (L) in blue.
136
Figure S6.8: LCMS Traces of Naturally Occurring Cycloamanide Core Regions Cyclized
by POPB. Substrate (S) signals are shown in green, cyclized core domain (C) in red, and
linearized core domains (L) in blue. Truncated 25mer peptide was the final product from the
phallacidin (PHA) substrate (signal in purple).
137
Figure S6.9: Simultaneous Production of Ten Cyclic Peptides Using POPB. The peptides
were all based on the α-amanitin core sequence and correspond to those shown in Supplementary
Figures 6.1-6.7. Shown are overlaid EICs for their expected masses.
138
Figure S6.10: Batch Production of Cyclic Peptides Using POPB. In each batch, six to eight E.
coli strains expressing different 35mer precursor peptides were grown and processed en masse
through POPB treatment. Sequences numbered were observed in the EICs; sequences encoded in
pink were not seen.
139
Figure S6.10 (cont’d)
140
Figure S6.10 (cont’d)
141
Figure S6.10 (cont’d)
142
Figure S6.10 (cont’d)
143
Table S6.2: Alphabetical List of Cyclic Peptides Produced with POPB.
Individual Assays Individual Assays Library Library
AWGIGCNP IWGISCNP KWGVACNP VWATACRP
AWLA(D-Thr)CP IWGLGCNP LWAQGCYP VWGCGCGP
FFVPPAFFPP IWGNGCNP LWGFACGP VWGIACTP
I(5-hydroxyTrp)GIGCNP IWGSGCNP LWGLACQP VWGPGCVP
IAGIGCNP IWLIGCNP LWGMGCWP VWGRGCQP
IFGIGCNP IWNIGCNP LWGSGCSP WWAGACLP
INGIGCNP IWSIGCNP LWGVACPP WWGCACLP
ISGIGCNP LWGIGCNP MWGMACFP WWGGGCRP
IW(N-methylAla)IGCNP MLGFLPLP NWGGACSP YWASACAP
IW(β-Ala)IGCNP MLGFLVLP NWGLACGP YWAYGCVP
IWAIGCNP NWGIGCNP QWGAACLP YWGQGCSP
IWGAGCNP SWGIGCNP QWGRGCLP
IWGAGIGAGCNP Library RWANACLP
IWGAVSGIGAVSGCNP AWAAGCSP RWGHACYP
IWGGIGGCNP AWADGCRP SWACGCSP
IWGIACNP AWGSGCSP SWAHGCHP
IWGIGANP AWGVGCMP SWAIACLP
IWGIGCAP CWALGCFP SWALGCVP
IWGIGCDP CWAVACAP SWASGCLP
IWGIGCLP CWGGGCQP SWGAGCEP
IWGIGCN(4-hydroxyPro) FWGSACFP SWGQACIP
IWGIGCNP FWGTGCFP SWGQGCHP
IWGIGCQP GWGAACCP SWGTACVP
IWGIGCSP GWGFGCFP SWGTGCYP
IWGIGGCNP HWGHACVP TWGAGCQP
IWGIGLNP HWGSGCRP TWGGGCMP
IWGIGNNP IWAHACVP VWAFACAP
IWGIGSNP IWALACVP VWAFGCFP
IWGILCNP IWAYGCYP VWAMGCTP
IWGINCNP IWGWGWGP VWASACVP
144
Table S6.3: Alphabetical List of Peptides Not Efficiently Cyclized by POPB.
Individual Assays
AWLATCP
AWLVDCP
IWGIGCP
IWGIGP
SFFFPIP
VFFAGP
Library
DWAPACFP
DWARACSP
DWGSGCVP
EWAAACPP
HWGRGCLP
IWGEGCWP
LWACACKP
PWGPACHP
RWAAACAP
RWALACVP
RWATACKP
RWGCGCLP
RWGLACCP
SWARACVP
SWGRACKP
SWGRGCSP
WWAKGCYP
YWAIACNP
YWAQACGP
YWAVACTP
YWGVACAP
145
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