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Mechanisms of S-Adenosylmethionine
Radical Enzymes
Kristin Plessel
Reich Group
September 7, 2006
2
General Enzyme Catalysis
Transition state stabilization Lowers activation energy
Pauling L. Chem. Eng. News 1946, 24, 1375.
http://www.mie.utoronto.ca/labs/lcdlab/biopic/biofigures.htm
3
Enzyme Control of Reactive Radicals
Radicals are highly reactive intermediates Prone to undesirable side reactions
“Negative catalysis” Selectivity by preventing undesired reactions Lengthens lifetime of radical Reaction with relatively high barrier more likely
Enzymatic control Isolation of reactive intermediates from small molecule
quenchers Conformational control
Retey, J. Angew. Chem. Int. Ed. Engl. 1990, 29, 355-361.
4
SAM in Methylating Enzymes
Nu= proteins, DNA, RNA, phospholipids, carbohydrates,
polysaccharides and other small molecules
Chiang, P.K.; Gordon, P.K.; Tal, J.; Zeng, G.C.; Doctor, B.P.; Pardhasaradhi, K.; McCann P.P. FASEB J. 1996, 10, 471-480.
Cantoni, G.L. Annu. Rev. Biochem. 1975, 44, 435-451.
O
OH OH
N
N
NH2
N
N
S
OOC
+
NH3+
H3C
O
OH OH
N
N
NH2
N
N
S
OOC
NH3
+
Nu
S-5'-deoxyladenosyl-L-methionine(SAM)
S-5'-deoxyladenosyl-L-homocysteine(SAH)
NuCH3
Methylase
5
SAM in Radical Enzymes
O
OH OH
N
N
NH2
N
N
S
OOC
NH3+
H3C
CH2
+
Methionine(Met)
5'-deoxyladenosyl radical(Ado )
O
OH OH
N
N
NH2
N
N
S
OOC
+
NH3+
H3C
S-5'-deoxyladenosyl-L-methionine(SAM)
e
O
OH OH
N
N
NH2
N
N
CH2R-H
O
OH OH
N
N
NH2
N
N
H3C + R
Ado 5'-deoxyadenosine(AdoH)
6
The Radical SAM Enzyme Superfamily
Family identified in 2001 through iterative sequence profiling Includes over 600 postulated members Found in 126 species
Biochemical pathways DNA precursor, vitamin, cofactor, antibiotic, and herbicide
biosynthesis Various biodegradation pathways
Half have unknown reactivity
Sofia, H.J.; Chen, G.; Hetzler, B.G.; Reyes-Spindola, J.F.; Miller, N.E. Nucleic Acids Res. 2001, 29, 1097-1106.
7
The Radical SAM Enzyme Common Characteristics
Requires SAM and reductant for activity FeS cluster at the active site Generally active in anaerobic conditions Strictly conserved Cys-X-X-X-Cys-X-X-Cys motif
Sofia, H.J.; Chen, G.; Hetzler, B.G.; Reyes-Spindola, J.F.; Miller, N.E. Nucleic Acids Res. 2001, 29, 1097-1106.
8
Classes of Radical SAM Enzymes
Catalytic SAM Enzymes Adenosyl radical generates a substrate radical
Stoichiometric SAM Enzymes Non-Activase Radical SAM Enzymes
Adenosyl radical generates a substrate radical
Activase Radical SAM Enzymes Adenosyl radical generates a protein radical
9
Catalytic Radical SAM Enzymes
Lysine 2,3-Aminomutase(LAM)
Spore Photoproduct-lyase(SPP lyase)
N
HN
O
O
R
N
HN
O
O
R
N
NH
O
O
R
SPP lyase
N
NH
O
O
R
+
COO
NH3
H3N+
+LAM
COOH3N+
NH3+
10
Classes of Radical SAM Enzymes
Catalytic SAM Enzymes Adenosyl radical generates a substrate radical
Stoichiometric SAM Enzymes Non-Activase Radical SAM Enzymes
Adenosyl radical generates a substrate radical
Activase Radical SAM Enzymes Adenosyl radical generates a protein radical
11
Non-activase Stoichiometric Radical SAM Enzymes
O
S-ACP
LipAO
S-ACP
S S
Biotin synthase(BioB)
Coproporphyrinogen III oxidase(HemN)
Lipoyl Synthase(LipA)
Formylglycine synthase(AtsB)
HNNH
NH HN
COO
COOCOO
COO
HNNH
NH HN
COOCOO
HemN
O
NHHN
CH3 COO
BioB
O
NHHN
COOS
O
OH
NH
AtsB
O
NH
H O
12
Classes of Radical SAM Enzymes
Catalytic SAM Enzymes Adenosyl radical generates a substrate radical
Stoichiometric SAM Enzymes Non-Activase Radical SAM Enzymes
Adenosyl radical generates a substrate radical
Activase Radical SAM Enzymes Adenosyl radical generates a protein radical
13
Activase Radical SAM Enzymes
Pyruvate Formate Lyase(PFL)
Anaerobic Ribonucleotide Reductase Class III (NrdD)
Cobalamin Independent Glycerol Dehydrase (Gdh)
Benzylsuccinate Synthase(BSS)
HO
OH
OHGdh
OHO
NrdDOO
OHOH
BaseP
O
O
OP
O
O
OP
O
O
OO
O
OH
BaseP
O
O
OP
O
O
OP
O
O
O
CO2
O
+ CoA-SHPFL
S-CoA
O
HCO2+
CH3
+
COOH
HOOC COOH
COOH
BSS(R)
14
Techniques
UV-Vis spectroscopy Isotopic labeling studies NMR spectroscopy Mass spectrometry Crystallography DFT calculations EPR spectroscopy ENDOR spectroscopy
15
EPR Spectroscopy
Magnetic Field
Ab
so
rpti
on
De
riv
ati
ve
Electron Paramagnetic Resonance Detects spin of unpaired electron
Fixed microwave frequency Variable magnetic field
Hyperfine splitting Electron spin and nuclear spin interaction
Drago, R.S. Physical Methods for Chemists; Sauders College:Orlando, FL, 1992, 2nd Ed, pp 559-594.
Que, L.., Jr. Ed.; Physical Methods in Bioinorganic Chemistry; University Science: Sausalito, CA, 2000; pp. 121-171.
H D
16
ENDOR Spectroscopy
Electron Nuclear DOuble Resonance EPR detected NMR Coupling between electronic and nuclear spins
Strong radio frequencies Fixed microwave frequency Monitor EPR intensity
Observe hyperfine couplings Experimental vs. Theoretical Data
Estimate of distance between
nucleus and unpaired electron
Hoffman, B.M. Acc. Chem. Res. 2003, 36, 522-529
Drago, R.S. Physical Methods for Chemists; Sauders College:Orlando, FL, 1992, 2nd Ed, pp 594.
v-v(13C) (MHz)
17
Outline
Introduction Shared Mechanism
Formation of adenosyl radical Individual Mechanisms Conclusions
18
First Radical SAM Enzyme:Lysine-2,3-Aminomutase
C C
X H
C C
H XCOO
NH3
H3N+
+LAM
COOH3N+
NH3+
Lysine 2,3-aminomutase (LAM)
Chirpich, T.P.; Zappia, V.; Costilow, R.N.; Barker, H.A. J. Biol. Chem. 1970, 245, 1778-1789.
Frey, P.A. FASEB J. 1993, 7, 662-670.; Marsh, E.N.G.; Patwardhan, A.; Huhta, M.S. Bioorg. Chem. 2004, 32, 326-340.
~30 kcal/mol
X= N, O, C
SAM Vitamin B12
+ reductant≥60 kcal/mol
19
Second Radical SAM Enzyme:Pyruvate Formate Lyase
Activated with: PFL- Activating Enzyme (PFL-AE) SAM Reductant
Fe-S cluster present in PFL-AE
Knappe, J.; Neugebauer, F.A.; Blaschkowski, H.P.; Ganzler, M. Proc. Natl. Acad. Sci., U.S.A. 1984, 81, 1332-1335.
Broderick, J.B.; Duderstadt, R.E.; Fernandez, D.C.; Wojtuszewski, K.; Henshaw, T.F.; Johnson, M.K. J. Am. Chem. Soc. 1997, 119, 7396-7397.
Pyruvate Formate Lyase
CO2
O
+ CoA-SHPFL
S-CoA
O
HCO2+
20
Evidence of a Radical in a Radical SAM Enzyme: PFL
with SAM
without SAM
Knappe, J.; Neugebauer, F.A.; Blaschkowski, H.P.; Ganzler, M. Proc. Natl. Acad. Sci., U.S.A. 1984, 81, 1332-1335.
H H
PFL-Gly
SAMMetAdoH
PFL-Gly
H
PFL-AE
EPR spectra of PFL with PFL-AE
21
Proposed Shared Mechanism
SAM
Methionine
5’-Deoxyadenosine
22
Fe-S Cluster and SAM
EPR of [4Fe-4S]+ in PFL-AE changes in presence of SAM
Walsby, C.J.; Hong, W.; Broderick, W.E.; Cheek, J.; Ortillo, D.; Broderick, J.B.; Hoffman, B.M.
J. Am. Chem. Soc. 2002, 124, 3143-3150.
without SAM
withSAM
FeS
SFe
SS
FeFe
O
OH OH
N
N
NH2
N
N
S
OOC
+
NH3+
CH3
FeS
SFe
SS
FeFe
O
OHOH
N
N
NH2
N
N
S+
NH3
+
H3C
O
O++
23
SAM Coordination to Fe-S cluster
ENDOR Active state: [4Fe-4S]+
17O and 15N direct coordination to Fe
13C-Fe distance 3.3 ± 0.1 Å
17O
13C
14N 15N
Walsby, C.J.; Hong, W.; Broderick, W.E.; Cheek, J.; Ortillo, D.; Broderick, J.B.; Hoffman, B.M.
J. Am. Chem. Soc. 2002, 124, 3143-3150
SAM
O
O
S
Ado
N
C
+
+
H3
24
SAM Coordination to Fe-S cluster
Layer, G.; Moser, J.; Heinz, J.W.; Jahn, D.; Schubert, W.D. EMBO J. 2003, 22, 6214-6224.
HemN
25
SAM Coordination to Fe-S cluster
Layer, G.; Moser, J.; Heinz, J.W.; Jahn, D.; Schubert, W.D. EMBO J. 2003, 22, 6214-6224. Hänzelmann, P.; Schindelin, H.
Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 12870-12875. Berkovitch, F.; Nicolet, Y.; Wan, J.T.; Jarrett, J.T.; Drennan, C.L. Science 2004,
303, 76-79. Lepore, B.W.; Ruzicka, F.J.; Frey, P.A.; Ringe, D. Proc. Natl. Acad. Sci., U.S.A. 2005, 102, 13819-13824.
BioB
MoaA LAM
HemN
26
Catalytically Active Fe-S Cluster
PFL-AE, SAM
[4Fe-4S]+ 12 K
PFL-AE, SAM, PFL
Gly• 60 K
[4Fe-4S]2+
[4Fe-4S]+ 30
10
5
2
1
0
Time (min)
No Gly•
Gly•
Photoreduction of Fe-S in PFL-AE with 5-deaza-riboflavin
1:1 [4Fe-4S]+:Gly• [4Fe-4S]+ is
catalytically active state
Henshaw, T.F.; Cheek, J.; Broderick, J.B. J. Am. Chem. Soc. 2000, 122, 8331-8332.
EPR Spectra
27
PFL-AE:Trapping Adenosyl Radical
Short peptides can be substrates for PFL-AE Trapping with dehydroalanine rather than glycine
Wagner, A.F.V.; Demand, J.; Schilling, G.; Pils, T.; Knappe, J. Biochem. Biophys. Res. Commun. 1999, 254, 306–310.
O
OH OH
N
N
NH2
N
N
O
HN
O
OH OH
N
N
NH2
N
N
O
HN
O
OH OH
N
N
NH2
N
N
O
HNH
28
LAM:Adenosyl Radical Analogue
Magnusson, O.T.; Reed, G.H.; Frey, P.A. Biochemistry 2001, 40, 7773-7782.
Magnusson, O.T.; Reed, G.H.; Frey, P.A. J. Am. Chem. Soc. 1999, 121, 9764-9765.
EPR
O
OH
Base
D
H
H
O
OH
Base
H
H
H DO
OH
Base
H
D
D
B C D
O
OH
Base
D
D
D
E
O
OH
Base
H
D
D D
F
O
OH
BaseS
OOC
NH3+ e
MetCH3
HH
+
O
OH
Base
H
H
H
A
29
Proposed Mechanism
Walsby, C.J.; Ortillo, D.; Yang, J.; Nnyepi, M.R.; Broderick, W.E.; Hoffman, B.M.; Broderick, J.B. Inorg. Chem. 2005, 44, 727-741
SAM
30
Outline
Introduction Shared Mechanism of Radical SAM Enzymes Individual Mechanisms of Radical SAM Enzymes
Pyruvate Formate Lyase Lysine 2,3-Aminomutase Spore Photoproduct Lyase
Conclusions
31
Pyruvate Formate Lyase (PFL)
Anaerobic counterpart to pyruvate dehydrogenase in metabolism of glucose to acetyl CoA in Escherichia coli
Gly734, Cys419, Cys418 necessary for catalysis
Knappe, J.; Blaschkowski, H.P. Methods Enzymol. 1975, 41B, 508-517. Wagner, A.F.V.; Frey, M.; Neugebauer, F.A.; Schafer, W.; Knappe, J. Proc. Natl. Acad. Sci., U.S.A. 1992, 89, 996-1000
CO2
O
+ CoA-SHPFL
S-CoA
O
HCO2+
CO2
O+ SH
Cys418
S
Cys418
HCO2+
O
S
Cys418
O
+ CoA-SH
SH
Cys418S-CoA
O
+
32
Stability of Glycyl Radical
Radical on Gly734 Half life
~10 sec at rt in air ≥ 24 hr at rt in glovebox
Captodative effect X-ray structure of PFL
Gly734 buried in protein structure, less accessible to small molecule quenchers
PFL-“Deactivase” enzyme, Alcohol Dehydrogenase AdhE, safely quenches radical
Walsby, C.J.; Ortillo, D.; Yang, J.; Nnyepi, M.R.; Broderick, W.E.; Hoffman, B.M.; Broderick, J.B. Inorg. Chem. 2005, 44, 727-741Becker, A.; Kabsch, W. J. Biol. Chem. 2002, 277, 40036-40042.
Kessler, D.; Herth, W.; Knappe, J. J. Biol. Chem. 1992, 267, 18073-18079.
O
OHN
H
NH
33
PFL: D2O exchange of Gly734•
EPR: α-H of Gly734 radical shows exchange with D2O Site-directed mutagenesis shows reaction is facilitated by
Cys419, not Cys 418
Wagner, A.F.V.; Frey, M.; Neugebauer, F.A.; Schafer, W.; Knappe, J. Proc. Natl. Acad. Sci., U.S.A. 1992, 89, 996-1000.Parast, C.V.; Wong, K.K.; Lewisch, S.A.; Kozarich J.W. Biochemistry, 1995, 34, 2393-2399.
NH
R
O
R
Gly734
SR
D
Cys419
H
NH
R
O
R
Gly734
SR
Cys419
NH
R
O
R
Gly734
SR
Cys419
NH
R
O
R
Gly734
SR
H
Cys419
DDH
34
pyruvate734Gly
419Cys SH
418Cys SH
734GlyH
419Cys S
418Cys SH
O
O
O734GlyH
419Cys
418Cys
SO
O
O
734GlyH
419Cys
SH418Cys
S O
O
O
formate
734Gly
419Cys SH
418Cys S O
CoA
Acetyl CoA
SH
734Gly
419Cys
SH418Cys
S O
PFL: 1st Half of ReactionKozarich Proposed Mechanism
Brush, E.J.; Lipsett, K.A. Kozarich, J.W. Biochemistry 1988, 27, 2217-2222.Parast, C.V.; Wong, K.K.; Lewisch, S.A.; Kozarich J.W. Biochemistry, 1995, 34, 2393-2399.
Bernardi, R.; Caronna, T.; Galli, R.; Minisci F.;.Perchinunno M. Tetrahedron Lett. 1973, 14, 645-64.
35
pyruvate734Gly
419Cys SH
418Cys SH
formate
CoA
Acetyl CoA
734GlyH
419Cys
418Cys
SOH
O
O
S
734GlyH
419Cys
418Cys
S OH
O
OS
734GlyH
419Cys
418Cys
S O
O
OHS
734GlyH
419Cys
S418Cys
S O
O
OH
734GlyH
419Cys
S418Cys
S O
O
OH
734GlyH
419Cys
418Cys
S O
S
734Gly
419Cys
418Cys
S O
SH
PFL: 1st Half of Reaction Knappe Proposed Mechanism
Knappe, J.; Elbert, S.; Frey, M.; Wagner, A.F.V. Biochem. Soc. Trans. 1993, 21, 731-734.
36
PFL: 1st Half of Reaction Crystal Structure
Becker, A.; Fritz-Wolf, K.; Kabsch, W.; Knappe, W.; Schultz, S.; Wagner, A.F.V. Nat. Struct. Biol. 1999, 6, 969-975.
37
PFL: 1st Half of Reaction Methacrylate Inhibition
Irreversible inhibition 14C labeled methacrylate confirmed consistent alkylation
of Cys418 Gly734• remains intact
Plaga, W.; Vielhaber, G.; Wallach, J.; Knappe, J. FEBS Lett. 2000, 466, 45-48.Lucas, M.F.; Ramos, M.J. J. Am. Chem. Soc. 2005, 127, 6902-6909.
pyruvate methacrylate
O
O
O
O
O
CH2
38
PFL: 1st Half of Reaction Methacrylate Inhibition
734GlyH
419Cys SH
418Cys SCH2
O
O
734GlyH
419Cys SH
418Cys S CH2
O
O
734GlyH
419Cys S
418Cys S CH2
O
O
H
Plaga, W.; Vielhaber, G.; Wallach, J.; Knappe, J. FEBS Lett. 2000, 466, 45-48.Lucas, M.F.; Ramos, M.J. J. Am. Chem. Soc. 2005, 127, 6902-6909.
734GlyH
419Cys SH
418Cys SO
O
O
734GlyH
419Cys SH
418Cys SO
O
O
734GlyH
419Cys SH
418Cys S O
O
O
734GlyH
419Cys S
418Cys S O
O
OH
39
pyruvate734Gly
419Cys SH
418Cys SH
734GlyH
419Cys S
418Cys SH
734GlyH
419Cys SH
418Cys S O
O
O
734GlyH
419Cys SH
418Cys SO
O
O
734GlyH
419Cys SH
418Cys S O
O
O
734GlyH
419Cys S
418Cys S O
O
OH
formate
734GlyH
419Cys S
418Cys S O
734Gly
419Cys SH
418Cys S O
CoA
Acetyl CoA
PFL: 1st Half of Reaction Currently Accepted Mechanism
Frey, P.A.; Hegeman, A.D.; Reed, G.H. Chem. Rev. 2006, 106, 3302-3316.
40
PFL: 2nd Half of Reaction Polar Mechanism
Conventional acyl transfer by nucleophilic attack with radical bystander
Himo, F.; Eriksson, L.E. J. Am. Chem. Soc. 1998, 120, 11449-11455.
734Gly
419Cys SH
418Cys S O
S-CoA
734Gly
419Cys SH
418Cys SO
S-CoA
O
S-CoA
734Gly
419Cys SH
418Cys S
734Gly
419Cys SH
418Cys SH
41
PFL: 2nd Half of Reaction Radical Mechanism H atom transfer to form CoAS• by followed by homolytic acyl
transfer Radical acyl protein 105 fold more reactive than non-radical
Wong, K.K.; Kozarich, J.W. Metal Ions in Biol. Sys. 1994, 30, 279-313. Guo, J.D.; Himo, F. J. Phys. Chem. B 2004, 108, 15347-15354.
O
S-CoA
734Gly
419Cys SH
418Cys S O
HS-CoA
734GlyH
419Cys S
418Cys S O
HS-CoA
734GlyH
419Cys SH
418Cys S O
S-CoA
734GlyH
419Cys SH
418Cys SO
S-CoA734GlyH
419Cys SH
418Cys SAcS-CoA
734GlyH
419Cys S
418Cys SH
734Gly
419Cys SH
418Cys SH
42
Outline
Introduction Shared Mechanism of Radical SAM Enzymes Individual Mechanisms of Radical SAM Enzymes
Pyruvate Formate Lyase Lysine 2,3-Aminomutase Spore Photoproduct Lyase
Conclusions
43
Lysine 2,3-Aminomutase (LAM)
O
OH
N
O3PO
H
2
First step in metabolism of lysine in Clostridia Stereospecific reaction Catalytic SAM and pyridoxal phosphate (PLP)
Chirpich, T.P.; Zappia, V.; Costilow, R.N.; Barker, H.A. J. Biol. Chem. 1970, 245, 1778-1789.
COO
NH3
H3N+
+
LAMH
HHCOO
H
H3N+
+
H
HH3N
44
LAM:Tritium Transfer Experiments
Baraniak, J.; Moss, M.L.; Frey, P.A. J. Biol. Chem. 1989, 264 1357-1360
COO
NH3
H3N+
LAM
COO
NH3
H3N+
COOH3N+
NH3+
3H 3H
O
OH OH
N
N
NH2
N
N
S
OOC
+
NH3+
CH3 3H
O
OH OH
N
N
NH2
N
N
S
OOC
+
NH3+
CH33H
+ +
45
LAM:Proposed Mechanism
Baraniak, J.; Moss, M.L.; Frey, P.A. J. Biol. Chem. 1989, 264 1357-1360Danen, W.C.; West, C.T. J. Am. Chem. Soc. 1974, 96, 2447-2453.
N
OH
N
O3PO
Lys337
HN
OH
N
O3PO
H CH COOCH
H
+H3NCH2CH2CH2
Ado
lysine
HN
OH
N
O3PO
CH COOCH+H3NCH2CH2CH2
AdoH
HN
OH
N
O3PO
CH COOCH+H3NCH2CH2CH2
AdoH
HN
OH
N
O3PO
CH COOCH+H3NCH2CH2CH2
AdoH
HN
OH
N
O3PO
CH2 COOCH+H3NCH2CH2CH2
Ado
-lysine
2
22
2
2
2
46
H N
C
CH3
COOEtH2C
Br
Bu3SnH, AIBN H N
C
CH3
COOEtH2C
H N
C
CH3
COOEtH2C
H N
C
CH3
COOEtH3C
H N
C
CH3
COOEtH2C
H
LAM: Role of PLP Chemical Model System
B:C1:13
65% yield
Han, O.; Frey, P.A. J. Am. Chem. Soc. 1990, 112, 8982-8983.
A B
C
47
LAM: Steady State Radical
COO
NH3
H3N+
+
COO
NH3
H3N+
+D
D
D D
DDD
D
Ballinger, M.D.; Reed, G.H.; Frey, P.A. Biochemistry 1992, 31, 949-953. Ballinger, M.D.; Frey, P.A.; Reed, G.H. Biochemistry 1992, 31, 10782-10789.
COO
NH3
H3N+
+D
COO
NH3
H3N+
+
13C
EPR
HN
OH
N
O3PO
CH COOCH+H3NCH2CH2CH2
2
48
LAM: Analogue Radicals4-Thia-L-lysine
Wu, W.; Lieder, K.W.; Reed, G.H.; Frey, P.A. Biochemistry 1995, 34, 10532-10537.
Miller, M.; Bandarian, V.; Reed, G.H.; Frey, P.A. Arch. Biochem. Biophys. 2001, 387, 281-288
COO
NH3
H3N+
+H
COOH3N+
+HH3N
S
NH4+ +
S
H3N+ SH +
O
COOH
49
LAM: Analogue Radicals4-Thia-L-lysine
SCOO
NH3
H3N
+
DD
COO
NH3
H3N+
+
13C
S
SCOO
NH3
H3N+
+
Wu, W.; Lieder, K.W.; Reed, G.H.; Frey, P.A. Biochemistry 1995, 34, 10532-10537.
EPR
HN
OH
N
O3PO
CH COOCH+H3NCH2CH2S
2
50
LAM: Analogue Radicalstrans-4-Dehydrolysine
Wu, W.; Booker, S.; Lieder, K.W.; Bandarian, V.; Reed, G.H.; Frey, P.A. Biochemistry 2000, 39, 9561-9570.
COO
N
H3N+
HH
CH-PLP
H
COO
N
H3N+
CH-PLP
H
H
Ado AdoH
51
LAM: Analogue Radicalstrans-4,5-Dehydrolysine
COO
NH3
H3N+
+
COO
NH3
H3N+
+D
COO
NH3
H3N+
+D
D
COO
NH3
H3N+
+D
D
D D
DD
COO
NH3
H3N+
+D
D
D
D D
D D
A
B
C
D
E
Wu, W.; Booker, S.; Lieder, K.W.; Bandarian, V.; Reed, G.H.; Frey, P.A. Biochemistry 2000, 39, 9561-9570.
EPR
52
LAM: Currently Accepted Mechanism
Frey, P.A.; Hegeman, A.D.; Reed, G.H. Chem. Rev. 2006, 106, 3302-3316.
N
OH
N
O3PO
Lys337
HN
OH
N
O3PO
H CH COOCH
H
+H3NCH2CH2CH2
Ado
lysine
HN
OH
N
O3PO
CH COOCH+H3NCH2CH2CH2
AdoH
HN
OH
N
O3PO
CH COOCH+H3NCH2CH2CH2
AdoH
HN
OH
N
O3PO
CH COOCH+H3NCH2CH2CH2
AdoH
HN
OH
N
O3PO
CH2 COOCH+H3NCH2CH2CH2
Ado
-lysine
2
22
2
2
2
53
LAM: Enzyme ControlENDOR Spectroscopy
Lees, N.S.; Chen, D.; Walsby, C.J.; Behshad, E.; Frey, P.A.; Hoffman, B.M. J. Am. Chem. Soc. 2006, 128, 10145-10154.
54
Outline
Introduction Shared Mechanism of Radical SAM Enzymes Individual Mechanisms of Radical SAM Enzymes
Pyruvate Formate Lyase Lysine 2,3-Aminomutase Spore Photoproduct Lyase
Conclusions
55
Spore Photoproduct-lyase (SPP lyase)
Endospores formed by bacteria under nutrient deficient conditions
Resistant to heat, toxic chemicals, UV irradiation SPP-lyase catalyzes the repair of methylene-bridged
thymine dimers formed in spore DNA by UV irradiation
Setlow, P. J. App. Microbiol. 2006, 101,514-525. Friedel, M.G.; Berteau, O.; Pieck, J.C.; Atta, M.; Ollagnier-de-Choudens, S.; Fontecave, M.; Carell, T. Chem. Commun., 2006, 445-447.
N
HN
O
O
R
N
HN
O
O
R
N
NH
O
O
R
Spore Photoproduct
SPP lyase
N
NH
O
O
R
UV irradiation
+
56
SPP lyase: Chemical Model System
C6 radical of spore photoproduct can undergo β-scission
Mehl, R.A.; Begley, T.P. Org. Lett. 1999, 1, 1065-1066.
85%
N
N
O
O
N
N
O
O
SPh
Bu3SnH, (Bu3Sn)2AIBN, PhH
N
N
O
O
N
N
O
O
N
N
O
O
(6) (6)
57
SAM
SPP lyase:Tritium Labeling Experiments
3H Transfer from C6 to SAM No 3H transfer from methyl Adenosyl radical abstract an H atom from C6 SAM formed reversibly
Cheek, J.; Broderick, J.B. J. Am. Chem. Soc. 2002, 124, 2860-2861
N
HN
O
O
R
N
NH
O
O
R3H
3H
(6)
N
HN
O
O
R
N
NH
O
O
R
3H3C
C3H2
58
SPP lyase:Proposed Mechanism
Guo, J.D.; Luo, Y.; Himo, F. J. Phys. Chem. B 2003, 107, 11188-11192.
59
Radical SAM Enzymes:Conclusions
Large family of over 600 postulated enzymes < 5% characterized
Shared mechanism for formation of adenosyl radical Independent and unique uses of adenosyl radical
Generate protein or substrate radical Diverse reactions
Many unique and powerful mechanisms yet to discover Novel radical chemistry
60
Acknowledgments Hans Reich
Ieva Reich
Practice Talk Attendees Melissa Boersma Seth Horne Luke Lavis Amanda King
Reich Group Kris Kolonko Amanda Jones
Perry Frey
Erin McElroy Katie Partridge Kim Peterson Kathy Van Heuvelen
Michael Mason
61
EXTRA SLIDES
62
PFL:Chemical Model Support
Minisci et al. reported cleavage of α-keto esters with Fenton’s reagent
O
COOEt COOEt
HO-O OHH2O2
COOEt
O OH O
OH+ COOEt
FeSO4
63
PFL:Mercaptopyruvate Inhibitor
64
PFLHypophosphite inhibitor
65
LAM: Analogue Radicalstrans-4,5-Dehydrolysine
Wu, W.; Booker, S.; Lieder, K.W.; Bandarian, V.; Reed, G.H.; Frey, P.A. Biochemistry 2000, 39, 9561-9570.
kform = 2.9 ± 0.6 min-1
[4Fe-4S]+1
kloss = 2.6 ± 0.4 min-1
Time (min)
Equ
iv of Organic R
adical
Equ
iv o
f [4
Fe-
4S
]+
COO
N
H3N+
CH-PLP
H
H
66
Reduction Potentials
-1.8-1.6-1.4-1.2-1.0-0.8-0.6-0.4-0.2V
PFL-AE [4Fe-4S]+
Nonenzymatictrialkyl
sulfonium
Colichman, E.L.; Love, D.L. J. Org. Chem. 1953, 18, 40-46.; Hinckley, G.T.; Frey, P.A. Biochemistry 2006, 45, 3219-3225.;
Frey, P.A. Personal Communication. Frey, P.A.; Hegeman, A.D.; Reed, G.H. Chem. Rev. 2006, 106, 3302-3316.
LAM [4Fe-4S]+ with SAM LAM [4Fe-4S]+
with SAM and lysine
0.27 V
Estimated SAM
Keq≈ 10-5
ln K = nE°/ 0.0257 at 25° C
E° = 0.27 V
67
Acetyl Coenzyme A
68
Mössbauer Spectroscopy
Monitors nuclear transitions from absorption of γ-rays Energy of γ-ray absorption changed by:
Quadrupole interactions Magnetic interactions Changes in electronic environment
γ-ray emitter
sample
detector
Drago, R.S. Physical Methods for Chemists; Sauders College:Orlando, FL, 1977; 2nd Ed, pp 626-645.
Solomon, E.I.; Lever, A.B.P., Eds.; Inorganic Electronic Structure and Spectrocopy; Wiley-Interscience: New York, NY, 1999; Vol. 1, pp 161-211.
69
Identification of Fe-S cluster in PFL-AE
[4Fe-4S] usually stabilized by 4 Cys Site-directed mutagenesis of CxxxCxxC Labile Fe-S cluster with site-differentiated cluster
Precedent in aconitase
Mössbauer Spectroscopy
FeS
SFe
SS
FeFe
Fe FeS
FeSS
S1+
2+
FeS
FeS
1+
FeS
SFe
SS
FeFe
1+/2+
Na2S2O4
Krebs, C.; Henshaw, T.F.; Cheek, J.; Huynh, B.H.; Broderick, J.B. J. Am. Chem. Soc. 2000, 122, 12497-12506.
Kennedy, M.C.; Kent, T.A.; Emptage, M.; Merkle, H.; Beinert, H.; Munck, E. J. Biol. Chem. 1984, 259(23), 14463-14471.
70
A Unique Iron Site
Krebs, C.; Broderick, W.E.; Henshaw, T.F.; Broderick, J.B.; Huynh, B.H. J. Am. Chem. Soc. 2002, 124, 912-913.
without SAM
with SAM
difference spectrum
71
EXAFS
Extended X-ray Absorption Fine Structure Structure information on amorphous samples Emitted core electron interacts with surroundings and
influences absorption of x-rays Matching experimental spectra with theoretical
Distance to and identity of neighboring atoms within 4-5 Å Coordination number of atom
Scott, R.A. Physical Methods in Bioinorganic Chemistry; University Science: Sausalito, CA, 2000; pp 465-504.
72
Methionine Coordination:Se EXAFS
LAM
PFL-AE
BioB
Se-C
Se-Fe
Nathaniel J. Cosper, N.J.; Booker, S.J.; Ruzicka, F.; Frey, P.A.; Scott, R.A. Biochemistry 2000, 39, 15668-15673.Cosper, M.M.; Cosper, N.J.; Hong, W.; Shokes, J.E.; Broderick, W.E.; Broderick, J.B.; Johnson, J.B.; Scott, R.A. Protein Sci. 2003, 12, 1573-1577.
O
OH OH
N
N
NH2
N
N
Se
OOC
+
NH3+
CH3
OOC
NH3+
Se
Se-Methionine
73
SAM Coordination:ENDOR Spectroscopy
PFL-AE Isotropic coupling indicated local orbital overlap Assigned to a dative interaction between sulfonium and
sulfide
Walsby, C.J.; Hong, W.; Broderick, W.E.; Cheek, J.; Ortillo, D.; Broderick, J.B.; Hoffman, B.M. J. Am. Chem. Soc. 2002, 124, 3143-3150.
+
74
Mechanistic Differences
Catalytic Enzyme: LAM
Stoichiometric Enzyme: PFL and BioB
Cosper, M.M.; Cosper, N.J.; Hong, W.; Shokes, J.E.; Broderick, W.E.; Broderick, J.B.; Johnson, J.B.; Scott, R.A. Protein Sci. 2003, 12, 1573-1577
75
EPR Spectroscopy
Hyperfine Splitting By neighboring nucleus
with nuclear spin Magnitude of splitting
depends on nucleus
Drago, R.S. Physical Methods for Chemists; Sauders College:Orlando, FL, 1992, 2nd Ed, pp 559-594.
Que, L.., Jr. Ed.; Physical Methods in Bioinorganic Chemistry; University Science: Sausalito, CA, 2000; pp. 121-171.
En
erg
y
Magnetic Field
MS MI
1/21/2
-1/2
-1/2 1/2
-1/2
H D
