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Aminopeptidase from Sphingomonas capsulata

Tony Byun‡, Maria Tang‡, Alan Sloma‡, Kimberly M. Brown‡, Chigusa Marumoto¶,

Mikio Fujii¶, and Alexander M. Blinkovsky‡§

From ‡Novozymes Biotech, Inc., 1445 Drew Avenue, Davis, CA 95616, and the ¶Foods Technology Center, Japan Tobacco Inc., 443-1, Shirayamado, Ohito-cho, Tagata-gun, Shizuoka 410-2318, Japan

Running title: Aminopeptidase from Sphingomonas

Correspondent footnote:

Dr. Alexander M. Blinkovsky

Novozymes Biotech, Inc.

1445 Drew Avenue

Davis, CA 95616

Phone: (530) 757-0826

FAX: (530) 758-0317

Internet e-mail: albl@novozymes.com

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on February 28, 2001 as Manuscript M010608200 by guest on January 31, 2018

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A novel aminopeptidase with unique substrate specificity was purified from a

culture broth of Sphingomonas capsulata. This is the first reported aminopeptidase

to demonstrate broad substrate specificity and yet release glycine and alanine with

the highest efficacy. On a series of pentapeptide amides with different N-terminal

amino acids, this enzyme efficiently releases glycine, alanine, leucine, proline, and

glutamate with the lowest turnover value of 370 min-1 for glutamate. At pH 7.5 (pH-

optimum) and 25oC, the kinetic parameters for alanine para-nitroanilide were

found to be kcat=7600 min-1and Km=14 mM. For alanine ββ -naphthylamide, they

were kcat=860 min-1 and Km=6.7 mM.

Polymerase chain reaction primers were designed based upon obtained

internal sequences of the wild-type enzyme. The subsequent product was then used

to acquire the full length gene from a S. capsulata genomic library. An open reading

frame encoding a protein of 670 amino acids was obtained. The translated protein

has a putative signal peptide that directs the enzyme into the supernatant. A search

of the amino acid sequence revealed no significant homology to any known

aminopeptidases in the available databases.

The rising interest, both basic and applied, in monoaminopeptidases (EC 3.4.11) from

different sources has led to the discovery of a number of enzymes that differ from each other in

cellular location, catalytic mechanism, and substrate specificity (1-3). The majority of bacterial

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monoaminopeptidases are intracellular or membrane-bound metalloenzymes (1). Based on

substrate specificity, bacterial monoaminopeptidases can be divided into two basic categories:

specific aminopeptidases, that release only a limited number of amino acids, and those that are

able to liberate a relatively broad spectrum of N-terminal amino acid residues (1).

Proline and glycine are among the most difficult residues for aminopeptidases to

hydrolyze due their unique structures. Proline is unusual due to its cyclic structure and glycine is

identified by the lack of a side chain. Nature has developed a family of enzymes that recognize

proline exclusively (4). A monoaminopeptidase that preferentially releases glycine with high

efficiency has not yet been described and thus would be of high interest.

In this report we describe a novel extracellular monoaminopeptidase from

Sphingomonas capsulata. This enzyme has a clear preference for N-terminal glycine and

alanine. Due to this characteristic, this monoaminopeptidase has the potential to

significantly enhance the degree of protein hydrolysis (5) when used as a supplement to

endoproteases and other exopeptidases.

EXPERIMENTAL PROCEDURES

Materials – Chemicals used as buffers and reagents were commercial products of at least

reagent grade. para-Nitroanilides of L-amino acids and peptide substrates were from Sigma

Chemical Co. or BACHEM California. Pentapeptide amides were synthesized at the Core

Laboratories (Louisiana State University). Sphingomonas capsulata strain IFO12533 was

purchased from the Institute for Fermentation, Osaka (Osaka, Japan). Whatman glass microfiber

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2.7 µm filter and Nalgene Filterware equipped with a 0.45 µm filter were used for filtering

buffers and supernatants. Protein purification was performed on a Pharmacia FPLC 1 device with

column supports and resins from the same. Ultrafiltration units (10 ml, 180 ml and 350 ml) and

membranes were from Amicon. The Tricine gels and PVDF membranes used in the peptide

separation and sequencing process were from Novex. The molecular weight of proteins was

estimated using Novex Multi-Mark pre-stained and Mark 12 SDS-PAGE markers.

Endoproteinase Glu-C (V8 Protease) was obtained from Boehringer Mannheim. Assays were

performed on THERMOmax microplate reader, Shimadzu spectrophotometer UV160U or

Hewlett Packard Series 1050 HPLC system with column supports from Vydac, Inc. The protein

sequencer used was from Applied Biosystems (Model 476A). The sequencing reagents were

purchased from Perkin Elmer/Applied Biosystems Division.

Purification of 66 kDa aminopeptidase - Cultivation of S. capsulata strain IFO

12533 was performed for 15 hours at 31°C, 250 rpm, and initial pH 7.45 in 1.5 liters of

medium composed per liter of 10 g of bactopeptone, 5 g of yeast extract, 3 g of NaCl, 2 g

of K2HPO4, 0.1 g of MgSO4· 7H2O, and 5 g of glucose (autoclaved separately).

The culture broth supernatant (approximately 1 liter) was obtained by initial

centrifugation followed by filtration using a Whatman glass microfiber and Nalgene

Filterware 0.22 µm filters consecutively. The filtrate was concentrated using an Amicon

Spiral Ultrafiltration System equipped with a PM 10 ultrafiltration membrane. The

sample was equilibrated with 10 mM phosphate pH 6.0 buffer until the conductivity and

1 The abbreviations used are: FPLC, Fast Performance Liquid Chromatography; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; HPLC, High Performance Liquid Chromatography; PMSF, phenylmethylsulfonyl fluoride, pNA, para-nitroanilide.

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pH were equal to the loading buffer, 50 mM MES pH 6.0. The filtered solution was

loaded onto a 24 x 390 mm column containing approximately 180 ml of SP-Sepharose

Fast Flow, pre-equilibrated with 50 mM MES pH 6.0 buffer. Protein with

aminopeptidase activity was eluted with 240 ml gradient from 0 to 0.2 M NaCl in 50 mM

MES pH 6.0 buffer. Fractions with enzymatic activity toward Ala-pNA were pooled,

desalted using a PM 10 membrane, and equilibrated with 20 mM phosphate pH 7.0

buffer.

The pooled solution was then loaded onto a 20 ml Pharmacia Mono Q Beads

column pre-equilibrated with 20 mM phosphate pH 7.0 buffer. Protein with

aminopeptidase activity did not bind to the column and was collected in the flow-through.

The flow-through was concentrated using a PM 10 membrane system as above, and the

pH adjusted to 6.0 with 70 mM acetate pH 4.0 buffer.

The concentrated flow-through was loaded onto a 1.0 ml Pharmacia Mono S

column which had been pre-equilibrated with 50 mM MES pH 6.0 buffer. The

aminopeptidase was eluted with a 60 ml gradient from 0 to 0.2 M NaCl in 50 mM MES

pH 6.0 buffer. The fractions with significant activity were then pooled, concentrated, and

equilibrated with 50 mM phosphate pH 7.0 buffer containing 0.5 M (NH4)2SO4 using a

PM 10 membrane as above.

Finally, the concentrated sample was loaded onto a Pharmacia Phenyl Superose

5/5 pre-packed 7 x 50 mm column pre-equilibrated with 50 mM phosphate pH 7.0 buffer

containing 0.5 M (NH4)2SO4. Protein with aminopeptidase activity was then eluted with

a 30 ml gradient from 0.5 to 0 M (NH4)2SO4 in 50 mM phosphate pH 7.0 buffer.

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Fractions containing aminopeptidase activity were analyzed by SDS-PAGE and then

pooled.

Periplasmic extraction of S. capsulata – A culture broth was grown in a 500 ml

shake flask under the conditions described above. A sample was centrifuged and the

whole cells separated from the supernatant. The harvested cells were then resuspended in

40 ml of 10 mM phosphate buffer pH 6.0, which contained 1 mg/ml of lysozyme and

0.1% triton X-100, and stirred for 2 hrs. The resulting sample was centrifuged and the

supernatant analyzed.

N-terminal and internal amino acid sequences of wild type aminopeptidase –

SDS-PAGE electrophoresis and transblotting were performed according to the Novex

instruction manual. Stained protein bands were excised and sequenced following the

instruction manual from Perkin Elmer/Applied Biosystems Division. Data was collected

and analyzed on a Macintosh IIsi using Applied Biosystems 610 Data Analysis Software.

To generate peptide fragments of the enzyme in order to obtain internal

sequences, the protein was cleaved with cyanogen bromide by reconstituting a dried

sample of the purified protein in 70% formic acid with a few crystals of cyanogen

bromide and incubating for 18 hours at room temperature in the dark. The peptide

fragments were separated by SDS-PAGE electrophoresis using a 10-20% Novex Tricine

gel and sequenced as described above. To generate additional peptide fragments, the

purified aminopeptidase was digested using Endoproteinase Glu-C (V8 Protease). A 200

µg sample of purified aminopeptidase was equilibrated in 0.125 M Tris-HCl pH 6.7 to a

final volume of 60 µl, and 7.5 µl of 0.125 M Tris-HCl pH 6.7 containing 2.5 % w/v of

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SDS was added. The sample was boiled for 2 minutes then allowed to cool to room

temperature. Then 10 µl of a 400 µg/ml solution of Endoproteinase Glu-C was added

and the resulting sample was incubated at 37 °C for 2 hrs. To this solution, 45 µl of

Novex 2X Tricine sample buffer was added. The peptide fragments were separated by

SDS-PAGE electrophoresis using a 10-20% Novex Tricine gel and sequenced as

described above.

Metal content analysis – The metal content was determined by atomic absorption

spectroscopy using a Perkin-Elmer 2380 Atomic Absorption Spectrophotometer equipped with

an HGA-400 Graphite Furnace. Each measurement was performed in triplicate. The

concentration was determined by running a standard curve of known metal concentration.

Physico-chemical characterization –

(i) Assay. Aminopeptidase activity was monitored using Ala-pNA as the

substrate. A stock solution of 100 mg/ml of Ala-pNA in dimethylsulfoxide was diluted

with 50 mM phosphate pH 7.5 buffer to a concentration of 2 mg/ml. The reaction of the

aminopeptidase with the para-nitroanilide was initiated when a 10-50 µl aliquot of the

enzyme solution was added to 200 µl of the substrate solution in a microtiter plate well.

Initial rates of hydrolysis of the para-nitroanilide were monitored at 405 nm at room

temperature using a THERMOmax microplate reader.

(ii) Substrate specificity and inhibition study. Stock solutions of para-

nitroanilides of various amino acids in dimethylsulfoxide (100 mg/ml) were diluted with

50 mM MOPS buffer pH 7.5 to concentrations of 2 mg/ml. Where the substrates were

incompletely soluble, their suspensions were used. The reaction of the S. capsulata

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aminopeptidase with each para-nitroanilide was initiated when an aliquot (10 µl) of the

enzyme solution in 50 mM phosphate pH 7.0 was added to 190 µl of a substrate solution

in a 96 well microtiter plate. Hydrolysis of the para-nitroanilides was monitored at 405

nm and 25°C.

Enzymatic hydrolysis of pentapeptides (see structures in Table 2) was performed

at pH 7.5 in 50 mM MOPS buffer at 21 °C. Concentrations of the peptides in the

incubation mixtures were between 1.10 and 1.14 mM. In order to stop the reactions, 50

µl aliquots of the incubation mixture were mixed with 50 µl of 0.1 M HCl. The resulting

samples were analyzed for free amino acids by reverse-phase HPLC (6). The

concentrations of the peptide solutions were determined using an extinction coefficient of

1440 M-1 cm-1 at 280 nm for the tyrosine residue.

The aminopeptidase was incubated for 2.5 hr in 50 mM MOPS buffer pH 7.5

which contained either no inhibitor, 1 mM EDTA, 1 mM o-phenanthroline, or 1 mM

PMSF. Following the incubation, the enzyme samples were assayed using Ala-pNA as

described above.

(iii) pH-Optimum. An aliquot (20 µl) of a stock solution of Ala-pNA in

dimethylsulfoxide (100 mg/ml) was diluted with 980 µl aliquots of sodium acetate-Tris-

HCl buffer (0.125 M) which had different values of pH between 5.0 and 8.5. The

resulting pH of the substrate solutions was measured. A stock solution (0.05 mg/ml )of

the aminopeptidase in 50 mM phosphate buffer was diluted 5-fold by 10 mM Tris-HCl

buffer pH 7.5. The reaction mixture contained 200 µl of the substrate solution and 10 µl

of an enzyme solution at room temperature.

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(iv) Temperature optimum. An aliquot (970 µl) of 50 mM MOPS buffer pH 7.5

was incubated for 15 minutes at the chosen temperature which was maintained using a

thermojacket (Shimadzu Cell positioner CPS-240A) of the spectrophotometer. Then, 30

µl of 100 mg/ml Ala-pNA in DMSO was added. The reaction was initiated by adding 7

µl of enzyme solution. Initial velocities were monitored over a 2 minute period at 405

nm.

(v) Thermal stability. An enzyme aliquot (10 µl) was added to 190 µl of 50 mM

phosphate buffer pH 7.5, which had been preincubated for 30 minutes at the chosen

temperature. The sample was placed on ice after a 20 minute incubation. The samples

were then assayed at room temperature following the protocol shown above.

(vi) Sequential release of N-terminal amino acid residues from a natural peptide.

Leucine Enkephalin was dissolved in 1 ml of 50 mM MOPS buffer, pH 7.5 to a final

concentration of 1 mg/ml. Enzymatic hydrolysis was initiated by 8.3 µg of S. capsulata

aminopeptidase. After incubation at room (21°C) temperature, aliquots of the incubation

mixture were added to 0.1 N HCl to terminate the reaction. The free amino acids of the

sample were analyzed by reverse-phase HPLC (6).

Cloning –

(i) Construction of genomic DNA library. Genomic DNA was isolated from S.

capsulata IFO 12533 using a QIAGEN Tip-500 column as per manufacturer’s

instructions (7). The library was constructed by ligating Sau 3A partially-digested (5-7

kb) S. capsulata IFO 12533 chromosomal DNA into the Bam HI sites of the vector

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pSJ1678 (8) (Figure 1) and transformed into Escherichia coli XL1 Blue MR

supercompetent cells (Stratagene, Inc.).

(ii) PCR amplification of aminopeptidase coding sequences. The following

primers were synthesized based on amino acid sequence data obtained from peptide

fragments obtained following cyanogen bromide and V-8 protease digestion of the

purified aminopeptidase.

Forward primer: 5’-GCRTCRTANGCRTCNCC-3’

Reverse primer: 5’-ACYTTYTCRTCYTTRTC-3’

(R=A or G, Y=C or T, N=A or G or C or T)

Amplification reactions were prepared in 50 µl volume with 50 pmol of forward and

reverse primers, 1 µg of S. capsulata IFO 12533 chromosomal DNA as template, 1X

PCR buffer (Perkin-Elmer), 200 µM each of dATP, dCTP, dGTP, and dTTP, and 0.5 U

of AmpliTaq Gold (Perkin-Elmer). Reactions were incubated in a Stratagene Robocycler

40 (Stratagene) programmed for 1 cycle at 95° C for 10 minutes, 35 cycles each at 95° C

for 1 minute, 44° C for 1 minute, and 72° C for 1 minute, and 1 cycle at 72° C for 7

minutes. The resulting product of ~190 bp was cloned into vector pCR2.1/TOPO as per

manufacturer’s instructions (Invitrogen, Inc.).

(iii) Identification of aminopeptidase clones. The genomic S. capsulata IFO

12533 library was screened by colony hybridization using a PCR generated probe with

the Genius Chemiluminescent System (Boehringer-Mannheim Corp.) as per

manufacturer’s instructions.

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(iv) DNA sequence analysis of S. capsulata IFO 12533 aminopeptidase gene.

DNA sequencing of two aminopeptidase containing clones, pMRT004.1-7 and

pMRT004.1-14, was performed with an Applied Biosystems Model 373A Automated

DNA Sequencer (Applied Biosystems, Inc.) on both strands using a) the Primer Island

Transposition method (Applied Biosystems, Inc.) as per manufacturer’s instructions and

b) primer walking technique using dye-terminator chemistry (9). Oligonucleotide

sequencing primers were synthesized by Operon Technologies, Inc.

RESULTS

Localization and purification of the native enzyme – The aminopeptidase was

obtained from the supernatant of S. capsulata IFO 12533. Periplasmic extraction of the

whole cells was also performed but the enzyme was not present in the extract. It is

evident from this result that the S. capsulata aminopeptidase is a secreted enzyme.

The purification led to a protein that migrated as a single band of 66 kDa on SDS-

PAGE (Figure 2).

Physico-chemical properties of the enzyme –A specific activity of 105 units/mg

was determined for Ala-pNA under the condition described above, assuming that the A280

of a 1 mg/ml solution of the aminopeptidase is 1.89. The theoretical extinction

coefficient of the enzyme was calculated based on the deduced protein sequence (10).

We were able to determine the kinetic parameters for Ala-pNA (kcat=7600±850

min-1 and Km=14±2 mM) and alanine β-naphthylamide (kcat=860±90 min-1 and

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Km=6.7±1.1 mM). However, the kinetic parameters for the other amino acid para-

nitroanilides and β-naphthylamides could not be accurately measured. This can be

attributed to a combination of large Km values, as well as, poor solubility of the synthetic

substrates. In terms of relative activity, the S. capsulata aminopeptidase preferably

hydrolyzes alanine para-nitroanilide. It also demonstrates high efficacy on para-

nitroanilides of leucine, methionine, glycine, aspartic and glutamic acids (Table 1).

The lower estimation of turnover numbers of the S. capsulata aminopeptidase on

a series of pentapeptide amides with different N-terminal amino acids are shown in Table

2. The enzyme exhibited the highest "turnover" on the pentapeptide amide with N-

terminal glycine, followed by alanine, leucine, glutamate, and proline.

A study of the hydrolysis of several natural peptides, catalyzed by the S.

capsulata aminopeptidase, revealed that the enzyme is capable of hydrolyzing a variety

of peptide bonds. Among the bonds most readily hydrolyzed was a Gly-Gly bond (Table

3). This is very unusual. The Gly-Gly bond is extremely resistant to enzymatic

hydrolysis, probably due to a lack of side chain groups on both the N-terminal and

penultimate amino acids. The S. capsulata aminopeptidase also hydrolyzed the peptides

YAGFL and EALELARGAIFQA- NH2 with obvious "bottlenecks" at phenylalanine

(Data not shown). It is important to stress that it also releases N-terminal proline (Table

2). This aminopeptidase, meanwhile, does not split off N-terminal amino acids with a

penultimate proline and thus possesses no proline aminopeptidase activity.

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Only o-phenanthroline demonstrated an inhibitory effect among the class-specific

inhibitors tested. In this case, the residual activity was found to be 4% of the initial.

Neither EDTA nor PMSF influenced the performance of the enzyme.

Certain inorganic anions, that form salts with zinc of low solubility, were found to

have inhibitory properties. Among the compounds examined were phosphate,

ferrocyanide, and iodate. The Ki values for these ions were determined to be 3.0 mM, 4.2

mM, and 11 mM, respectively. There is a direct correlation between the Ki value and the

solubility product of the particula r anion with zinc.

A plot of the relative activity of S. capsulata aminopeptidase in the hydrolysis of

Ala-pNA gave a typical bell-shaped pH-dependence with a sharp optimum at pH 7.2-7.4.

An incubation of the enzyme for 20 min at 45 and 50oC led to losses of 40% and 100%

activity, respectively. The optimal temperature for the activity was determined to be

43°C.

Sequencing of the wild type S. capsulata aminopeptidase – The N-terminal

sequence of the 66 kDa homogeneous protein was determined to be blocked. Digestion

of the protein with cyanogen bromide resulted in fragments with molecular weights of 42,

30, 17, 15, 10, 6, and 4 kDa. The N-terminal sequence of the 10 kDa fragment was

determined to be AVNGDAYDADKLKGAITNAKTGNPGAGRPI. The N-terminal

sequences of the other bands were inconclusive. The digestion with Endoproteinase Glu-

C resulted in the generation of peptides with molecular weights of 40, 30, 25, 22, 20, 17,

10, 6, 5, and 4 kDa. The sequence

FKDEPNPYDKARMADAKVLSLFNSLGVTLDKDGKV was obtained from the 22 and

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17 kDa peptide fragments. The other bands were either not sequenced or the results were

inconclusive. The obtained protein sequences did not demonstrate homology to any

known aminopeptidases.

Sequence analysis of the DNA encoding S. capsulata aminopeptidase – Screening

of the S. capsulata IFO 12533 genomic library produced five colonies that exhibited

strong hybridization signals with the probe. Two plasmids carrying the aminopeptidase

gene were sequenced, and one was confirmed to contain the entire aminopeptidase gene.

Sequence analysis of the plasmid containing the aminopeptidase gene revealed an open

reading frame of 2010 nucleotides, encoding a protein of 670 amino acids. The G+C

content of this open reading frame was 65%. Based on the rules of von Heijne (11), the

first 32 amino acids likely comprise a secretory signal peptide which directs the nascent

polypeptide into the periplasm.

The calculated molecular weight of the primary translation product determined

from the deduced amino acid sequence of the S. capsulata aminopeptidase was 70.6 kDa,

which is consistent with the estimation of 66 kDa based on the mobility of the purified

protein on SDS-PAGE. The zinc binding motif HEXXH, which is present in a number of

metallopeptidase families (12), was identified in the primary sequence (Figure 3). A

BLAST search of the S. capsulata aminopeptidase against known databases (EMBL,

SWISSPROT, GENBANK, and GENESEQ) revealed no significant homology to any

known aminopeptidases. The highest percent identity (23%) was observed with a

hypothetical 67 kDa protein from Synechocystis sp.

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The presence of one atom of zinc per the enzyme molecule was detected by

atomic absorption spectroscopy. This method also indicated four iron atoms per the

enzyme molecule in a homogeneous preparation of the S. capsulata aminopeptidase.

There were no detectable levels of cobalt or calcium.

DISCUSSION

Bacteria hydrolyze different proteins in order to acquire essential amino acids

from the pool of free amino acids and peptides. Certain organisms, such as the

nutritionally fastidious Lactococcus lactis, apply a non-direct mechanism and employ a

cascade of endo- and exopeptidases in order to release N-terminal glycine (1, 13).

Others, such as Xanthomonas citri, have developed a more rational method, producing

aminopeptidases with broad substrate specificity (14). In this report, we show that S.

capsulata secretes a unique enzyme that preferably liberates N-terminal glycine. Due to

a combination of large Km values for both para-nitroanilides and β-naphthylamides

coinciding with low solubilities for these compounds, it is impossible to carry out a

comprehensive study of the substrate specificity of the S. capsulata aminopeptidase

utilizing kinetic data for these artificial substrates. Nonetheless, a few remarks can be

made: this enzyme apparently discriminates similar amino acids effectively releasing

leucine but not isoleucine or valine (Table 1); results for alanine para-nitroanilide and β-

naphthylamide unequivocally show that the structure of the leaving group for a substrate

affects both kcat and Km. Taking this into account and expecting that the substrate

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preference of an aminopeptidase toward derivatives of amino acids, such as para-

nitroanilides, and na tural peptides can be substantially different (15), the study of the

hydrolysis of non-protected peptides catalyzed by the S. capsulata aminopeptidase was

warranted. Comparative analysis of the Pro-pNA and Pro-Ala-Pro-Tyr-Lys-NH2

highlights the misleading role of the para-nitroanilide group. Apparently, it hinders the

binding of at least some amino acid residues, for example proline, by the enzyme. More

importantly, the S. capsulata aminopeptidase demonstrates an unusual substrate pattern

with the order of preference Gly>Ala>Leu in terms of relative activity (Table 2). A

plausible explanation for these features could be a catalytic pocket that is not deep and

exhibits very limited flexibility.

Leucine aminopeptidases are widely distributed in bacteria (1). Normally they are

completely passive toward glycine (16). There are a few bacterial aminopeptidases

described in the literature that demonstrate a reasonable ability to release alanine. The

alanine-specific aminopeptidase N from E. coli (17) was not shown to release N-terminal

glycine. The bimolecular constant for the thiol aminopeptidase from X. citri was almost

40-fold greater for alanine β-naphthylamide in comparison to glycine β-naphthylamide

(14). It is highly unlikely that this enzyme is capable of cleaving a Gly-Gly bond.

Aminopeptidase from S. capsulata occupies a unique niche among bacterial

proteases. This is the first reported enzyme that hydrolyzes natural peptides with

bimolecular constant values that are similar for glycine and alanine, or probably even

higher for glycine. This extraordinary substrate preference is undoubtedly exhibited in

the hydrolysis of leucine enkephalin catalyzed by the S. capsulata aminopeptidase (Table

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3). A quick and complete release of tyrosine and both glycine residues from this peptide

was observed after a 1.0 hr reaction, yet the Phe-Leu bond was nearly untouched. This

clearly demonstrates the substantially higher catalytic efficacy of this enzyme for amino

acids with small rather, than large, side chains. A high kcat value, at least 5400 min-1, for

releasing glycine is also uncommon. Another interesting feature of the S. capsulata

aminopeptidase is that, for an unknown reason, it is able to distinguish between similar

amino acid residues, like tyrosine and phenylalanine, in the case of non-protected

peptides (Table 3).

Metallo-aminopeptidases are predominant in bacteria (1). There are several

indirect indications that this is a zinc metalloenzyme. First, effective inhibition of the S.

capsulata aminopeptidase by o-phenanthroline, but not by PMSF or p-

chloromercuribenzoic acid, was observed. The enzyme is also inhibited by certain

anions, whose zinc salts have low solubility product value. (We assume that EDTA,

another strong chelator of transition metals, shows practically no inhibitory effect due to

its voluminous structure and polyanionic nature resulting in its inability to penetrate close

enough to the zinc of the catalytic site). A neutral pH-optimum (18) for the S. capsulata

aminopeptidase and a putative zinc binding domain HEXXH (15) in its amino acid

sequence are both indications of a zinc metalloenzyme.

Atomic absorption spectroscopy confirms the presence of one atom of zinc per

molecule of enzyme. In addition, four atoms of iron were also detected. A plausible

explanation might be non-specific binding of this metal to the protein molecule.

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The results that have been presented prove the scientific novelty of the S.

capsulata aminopeptidase. We believe that this enzyme will receive significant attention

from the food industry as well. It has an "industrial" pH-optimum, high specific activity,

and great performance in releasing alanine and glycine, two amino acids that give

considerably strong sweetness (19), in natural peptides.

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Acknowledgements

We thank Amy Palmer from the Department of Chemistry, Stanford University

for providing atomic absorption spectroscopy analysis.

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Biophys. Acta, in press.

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16. Gonzales, T.. and Robert-Baudouy, J. (1996) FEMS Microbiol. Rev. 18, 319-344, references

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Figure legends.

Fig. 1. Vector pSJ1678.

Fig. 2. SDS-PAGE of purified S. capsulata aminopeptidase stained with Coomassie

Brilliant Blue R-250. Left lane: molecular weight marker (From top to bottom) 200,

116.3, 97.4, 66.3, 55.4, 36.5, 31, 21.5 and 14.4 kDa, respectively; Right lane: purified S.

capsulata aminopeptidase

Fig. 3. The gene and the protein sequence.

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Table 1. Relative activity of the aminopeptidase with para-nitroanilides of different

amino acids (MOPS buffer, 0.125 mM, pH 7.5. [E]o=0.0194µM. Concentration of the

substrates is 7.7 mM).

Amino acid para-nitroanilide Relative activity (%)

Ala 100

Leu 27

Met 24

Gly 14

Asp 14

Glua 12

Lys 3

Pro 0

Ile 0

Val 0

Phea 0

aSolubility of para-nitroanilides of glutamate and phenylalanine is lower than 7.7 mM.

Their suspensions were used.

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Table 2. Substrate specificity of recombinant Sphingomonas capsulata aminopeptidase

expressed in Bacillus subtilis toward XAPYK-amide pentapeptides (for conditions, see

"Materials and methods").

______________________________________________________________________

X of XAPYK-amide turnovera (min-1)

______________________________________________________________________

Gly- 5400

Ala- 4100

Leu- 760

Glu- 580

Pro- 370

_______________________________________________________________________

aTurnover was calculated based on an initial velocity of the enzymatic hydrolysis of the

pentapeptides and is not equivalent to kcat in this case. It can be considered only as a

lower estimation of kcat. Because we do not know values of Km, the concentration of

peptides (1.10-1.14 mM) may be insufficient to provide maximal velocity.

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Table 3. Time release of N-terminal amino acids from Leucine Enkephalin (2.50 nmoles) by

recombinant S. capsulata aminopeptidase expressed in B. subtilis. For the specific conditions,

see Experimental Procedures section on the Physico-chemical characterization (VI).

Substrate Released Amount of released amino acid (nmol)

amino acid after incubation for:

_________________________________________________________

0 h 0.5 h 1.0 hr 3.0 h

Leucine Tyr 0 2.46 2.46 2.46

Enkephalina Gly 0 4.04 5.10 4.70

Phe 0 0.08 0.10 0.47

Leu 0 0.11 0.09 0.43

aThe sequence of Leucine Enkephalin is Tyr-Gly-Gly-Phe-Leu.

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pSJ1678(4679 bp)

kanD

cat

rep

p15a ori

EcoRI (1)NruI

ClaI HindIIIPstI

BamHIXmnI

NsiI

BglII1000

BamHIPstIHindIII

ClaI

NruI

BglII

SphI

2000XmnINdeINcoI

XmnI

3000

BglII

NsiI

4000

NcoI

XmnI

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CAGGTGCAGCCGGCGAGCAACAGCCGCCCGATGGCAGTGCCGATCGCTCATGGGGTGCCCGATGCGCAGG 70 Q V Q P A S N S R P M A V P I A H G V P D A Q ACGTGCCCTATCCCGGCACGATCGGGCTGCAGATCGATGCCACCGATCTGGCCACCGGGGCGTTCCGGGT 140 D V P Y P G T I G L Q I D A T D L A T G A F R V GGTGGAAACCGTGCCGGTGGCGGCCGATGCCAAGGAACTGATCCTGCAACTGCCGGCCTGGCTGCCGGGT 210 V E T V P V A A D A K E L I L Q L P A W L P G GAGCATGGCAATCGCGGCCCCGTGGCCGAGCTGGCCGGCATCACGTTTGAAGCCAAGGGCCAGAAGCTGG 280 E H G N R G P V A E L A G I T F E A K G Q K L CCTGGACCCGCGACCCGGTGGAAGTGAACGCGTTCCACATCCCCCTGCCCGCCGGCACCAGCGAAGTGGT 350 A W T R D P V E V N A F H I P L P A G T S E V V GGCCCGCTTCATCCACACCTCGCCGCTGCGCGACAGCGAAGGCCGCATCACCGTTACGCGCGAAATGCTC 420 A R F I H T S P L R D S E G R I T V T R E M L AACGTGCAGTGGGAGAAGATGAGCCTCTATCCCGCCGGTCACTATGTGCGGCAGATCAAGGTGCGTCCCA 490 N V Q W E K M S L Y P A G H Y V R Q I K V R P CCGTCAGCTTCCCGCAGGGTTGGACCGTGTTCACCGCGCTGGATGGCAAGACGCAGAGCGGCGCGGGCAA 560 T V S F P Q G W T V F T A L D G K T Q S G A G N TACCGTGACTTGGGCCGAAACCGACTATGAAACCCTGGTCGATTCGCCGATCTTTGCCGGGCTCTATGCC 630 T V T W A E T D Y E T L V D S P I F A G L Y A GCGCGGCATGATCTGGGCCACAACGTCTATTTCGATCTGGTGGCCGACAAGCCCGAGCTGCTGGCGATCA 700 A R H D L G H N V Y F D L V A D K P E L L A I AGCCGGAAAACCTGGCCGCCTATCGCAACCTGGCCGACGAAGCCGTGGGCGCATTCGGCGCGCGCCATTT 770 K P E N L A A Y R N L A D E A V G A F G A R H F CGATCACTACGATTTCCTGCTCGCGCTGACCGATCGCATGGGCAGCATCGGCCTGGAACACCACCGTTCC 840 D H Y D F L L A L T D R M G S I G L E H H R S AGCGAAAACCAGCAGGAACCCAAGAGCCTGACCGACTGGGCCGCCTATGACTGGGACCGCAACGTGATCG 910 S E N Q Q E P K S L T D W A A Y D W D R N V I CCCACGAATTCAGCCACAGCTGGGATGGCAAGTATCGCCGCTCGGCCAAGCTGTGGACGCCCGACTATCG 980 A H E F S H S W D G K Y R R S A K L W T P D Y R CCAGCCGATGCAGGACAACCTGCTGTGGGTCTATGAAGGGCAGACGCAGTTCTGGGGCCTGGTCCTGGCC 1050 Q P M Q D N L L W V Y E G Q T Q F W G L V L A GCACGCTCGGGCGTGCAGAGCAAGGACGTGGTCTTGGGCAGCCTCGCCAACTATGCCGGCACGTTCACCC 1120 A R S G V Q S K D V V L G S L A N Y A G T F T AGACCGCCGGGCGCGACTGGCGCTCGGTGGAAGACACGACGATGGATCCCATCTTCGCCGCCCGCAAGCC 1190 Q T A G R D W R S V E D T T M D P I F A A R K P CAAGCCCTATTCCTCGCTTACCCGTAACGAGGACTATTACACCGAAGGCGCGCTGGTGTGGCTGGAAGCG 1260 K P Y S S L T R N E D Y Y T E G A L V W L E A GACCAGATCATCCGCGATGGCACCGGCGGCAAGAAGGGCCTGGATGATTTCGCCAAGGCGTTCTTTGGCG 1330 D Q I I R D G T G G K K G L D D F A K A F F G TGCGCGACGGCGATTGGGGCGTGCTGACCTATGAATTCGATGACGTGGTCAAGACCCTCAACGGCGTCTA 1400 V R D G D W G V L T Y E F D D V V K T L N G V Y TCCCTATGACTGGGCCACGTTCCTCAAGACCCGCCTGCAGACGCCGGGCCAGCCGGTGCCGCTCGGCGGG 1470 P Y D W A T F L K T R L Q T P G Q P V P L G G ATCGAGCGCGGCGGCTACAAGCTGGAATTCAAGGACGAGCCCAACCCCTATGACAAGGCGCGCATGGCCG 1540 I E R G G Y K L E F K D E P N P Y D K A R M A ATGCCAAGGTGCTCAGCCTGTTCAACTCGCTGGGCGTGACGCTGGACAAGGACGGCAAAGTCACCGCCTC 1610 D A K V L S L F N S L G V T L D K D G K V T A S GCGCTGGGATGGCCCGGCGTTCAAGGCGGGGCTGGTTTCGGGCATGCAGGTGATGGCCGTGAACGGCGAC 1680 R W D G P A F K A G L V S G M Q V M A V N G D GCCTATGACGCGGACAAGCTCAAGGGCGCGATCACCAATGCCAAGACCGGCAACCCCGGCGCCGGCCGCC 1750 A Y D A D K L K G A I T N A K T G N P G A G R CGATCGAACTGCTGGTCAAGCGTGACGATCGCTTTGTCACGCTGCCGATCACCTATGCCGATGGCCTGCG 1820 P I E L L V K R D D R F V T L P I T Y A D G L R CTGGCCGTGGCTGGTGCGCACGGCGCCGGGCACGGCACCGACCGGGCTGGACAAGCTGCTGGCCCCGCAC 1890

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W P W L V R T A P G T A P T G L D K L L A P H GCCAGCAAGCTGCCCGTGGGCAAGGCTGCCAAG 1923 A S K L P V G K A A K

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Fujii and Alexander M. BlinkovskyTony Byun, Maria Tang, Alan Sloma, Kimberly M. Brown, Chigusa Marumoto, Mikio

Aminopeptidase from Sphingomonas capsulata

published online February 28, 2001J. Biol. Chem. 

  10.1074/jbc.M010608200Access the most updated version of this article at doi:

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