Transcript Mechanism of Radical SAM Enzymes

Mechanisms of
S-Adenosylmethionine
Radical Enzymes
Kristin Plessel
Reich Group
September 7, 2006
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
2
Enzyme Control of
Reactive Radicals

Radicals are highly reactive intermediates


“Negative catalysis”




Prone to undesirable side reactions
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.
3
SAM in Methylating Enzymes
NH2
NH2
N
N
Nu
NuCH 3
N
N
H3C
S
+
N
N
N
S
N
O
O
Methy lase
OOC
+
NH3
OOC
OH OH
S-5'-deoxyladenosyl-L-methionine
(SAM)
+
NH3
OH OH
S-5'-deoxyladenosyl-L-homocysteine
(SAH)
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.
4
SAM in Radical Enzymes
NH2
N
H3C
S
+
NH2
N
e
N
N
H3C
S
N
O
+
CH2
O
N
N
N
OOC
OOC
+ NH3
+ NH3
OH OH
NH2
NH2
CH2
O
N
OH OH
Ado
N
N
N
5'-deoxyladenosyl radical
(Ado )
Methionine
(Met)
S-5'-deoxyladenosyl-L-methionine
(SAM)
N
OH OH
R-H
H3C
N
N
N
O
+
R
OH OH
5'-deoxyadenosine
(AdoH)
5
The Radical SAM
Enzyme Superfamily

Family identified in 2001 through iterative sequence
profiling



Biochemical pathways



Includes over 600 postulated members
Found in 126 species
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.
6
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.
7
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
8
Catalytic Radical SAM
Enzymes
NH3+
+
H3N
LAM
COO
+
H3N
COO
+
NH3
Lysine 2,3-Aminomutase
(LAM)
O
HN
O
SPP ly ase
NH
N
N
R
R
O
O
O
O
HN
O
NH
+
N
N
R
R
O
Spore Photoproduct-lyase
(SPP lyase)
9
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
10
Non-activase Stoichiometric
Radical SAM Enzymes
COO
O
HN
O
BioB
NH
CH3
NH
COO
HN
NH
HN
HemN
NH
HN
NH HN
COO
NH HN
COO
S
Biotin synthase
(BioB)
O
COO
COO
COO
COO
Coproporphyrinogen III oxidase
(HemN)
OH
O
LipA
H
O
AtsB
S-ACP
S-ACP
S
S
Lipoyl Synthase
(LipA)
N
H
O
N
H
O
Formylglycine synthase
(AtsB)
11
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
12
Activase Radical
SAM Enzymes
HOOC
CH3
(R)
BSS
COOH
+
COOH
COOH
OH
Benzylsuccinate Synthase
(BSS)
O
O
O
O P O P O P O
O
O
O
Base
NrdD
O
OH
O
O
O
O P O P O P O
O
O
O
OH
HO
Gdh
O
Cobalamin Independent
Glycerol Dehydrase (Gdh)
Base
O
OH
O
Anaerobic Ribonucleotide
Reductase Class III (NrdD)
HO
OH
PFL
O
+ CoA-SH
CO2
+
HCO2
S-CoA
Pyruvate Formate Lyase
(PFL)
13
Techniques








UV-Vis spectroscopy
Isotopic labeling studies
NMR spectroscopy
Mass spectrometry
Crystallography
DFT calculations
EPR spectroscopy
ENDOR spectroscopy
14


Electron Paramagnetic Resonance
Detects spin of unpaired electron



Fixed microwave frequency
Variable magnetic field
Absorption
Derivative
EPR Spectroscopy
Magnetic Field
Hyperfine splitting

Electron spin and nuclear spin interaction
H
D
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.
15
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
v-v(13C) (MHz)
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.
16
Outline


Introduction
Shared Mechanism



Formation of adenosyl radical
Individual Mechanisms
Conclusions
17
First Radical SAM Enzyme:
Lysine-2,3-Aminomutase
NH3+
X
LAM
+H N
3
+H N
3
COO
COO
+
NH3
H
H
C C
X
C C
Lysine 2,3-aminomutase
(LAM)
X= N, O, C
~30 kcal/mol
+ reductant
≥60 kcal/mol
SAM
Vitamin B12
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.
18
Second Radical SAM Enzyme:
Pyruvate Formate Lyase
O
PFL
O
+ CoA-SH
CO2
+
HCO2
S-CoA
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.
19
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.
Evidence of a Radical in a
Radical SAM Enzyme: PFL
with SAM
H
H
SAM
Met
AdoH
PFL-AE
PFL-Gly
H
PFL-Gly
without SAM
EPR spectra of PFL with PFL-AE
Knappe, J.; Neugebauer, F.A.; Blaschkowski, H.P.; Ganzler, M. Proc. Natl. Acad. Sci., U.S.A. 1984, 81, 1332-1335.
20
Proposed Shared Mechanism
SAM
Methionine
5’-Deoxyadenosine
21
Fe-S Cluster and SAM

EPR of [4Fe-4S]+ in PFL-AE changes
in presence of SAM
NH2
N
without
SAM
NH2
with
SAM
N
N
CH3
S+
N
N
N
N
H3C
N
O
OOC
+
OH OH
Fe
S
Fe
S
O
OH
+
NH3
S
Fe
+
S
+
S
Fe
Fe
S
Fe
O
S
S
Fe
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.
+
NH3
O
S
Fe
OH
22
SAM Coordination
to Fe-S cluster

ENDOR

17O
Active state:
[4Fe-4S]+
O
+S
C
+ N H3
 17O

and 15N direct
coordination to Fe
13C-Fe distance 3.3 ± 0.1 Å
Ado
SAM
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
23
O
SAM Coordination
to Fe-S cluster
HemN
Layer, G.; Moser, J.; Heinz, J.W.; Jahn, D.; Schubert, W.D. EMBO J. 2003, 22, 6214-6224.
24
SAM Coordination
to Fe-S cluster
HemN
BioB
MoaA
LAM
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.
25
Catalytically Active
Fe-S Cluster
Time (min)
[4Fe-4S]+
EPR Spectra
30

Gly•
10


5
2
[4Fe-4S]2+
1
0
No Gly•
PFL-AE, SAM
PFL-AE, SAM, PFL
[4Fe-4S]+ 12 K
Gly• 60 K
Photoreduction of
Fe-S in PFL-AE
with 5-deazariboflavin
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.
26
PFL-AE:
Trapping Adenosyl Radical


Short peptides can be substrates for PFL-AE
Trapping with dehydroalanine rather than glycine
NH2
N
O
N
O
OH OH
N
N
HN

NH2
HN

N
O
N
NH2
N
N
N
H
HN
O
O
OH OH
N
N
N
O
OH OH
Wagner, A.F.V.; Demand, J.; Schilling, G.; Pils, T.; Knappe, J. Biochem. Biophys. Res. Commun. 1999, 254, 306–310.
27
LAM:
Adenosyl Radical Analogue
EPR
+ CH
3
S
OOC
+
NH3
Met
Base
e
O
H
Base
H
O
H
H
H
OH
OH
A
Base
H
H
H
OH
D
H
B
D
O
H
D
Base
Base
O
O
D
C
D
Base
D
O
Base
D
D
O
D
D
D
OH
E
OH
H
OH
OH
H
F
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.
28
Proposed Mechanism
SAM
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
29
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
30
Pyruvate Formate Lyase (PFL)
O
PFL
+
O
CoA-SH
CO2
+
HCO2
+
HCO2
S-CoA
O
O
+
CO2
SH
S
Cy s418
Cy s418
O
O
S
Cy s418
+
CoA-SH
SH
+
S-CoA
Cy s418

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
31
Stability of Glycyl Radical


Radical on Gly734
Half life




O
H
N
H
Captodative effect
X-ray structure of PFL


~10 sec at rt in air
≥ 24 hr at rt in glovebox
O
H
N
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, 727-741
Becker, A.; Kabsch, W. J. Biol. Chem. 2002, 277, 40036-40042.
Kessler, D.; Herth, W.; Knappe, J. J. Biol. Chem. 1992, 267, 18073-18079.
32
PFL:
D2O exchange of Gly734•
Cy s419
R
S
Cy s419
R
D S
H
R
N
H

D
H D
R
O
Gly 734

Cy s419
R
H S
R
N
H
R
R
O
Gly 734
R
N
H
O
Gly 734
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.
33
PFL: 1st Half of Reaction
Kozarich Proposed Mechanism
734
Gly
pyruvate
734
734
Gly H
Gly H
O
O
O
O
419
Cy s
419
Cy s
SH
S
O
419
Cy s S
O
418
Cy s
418
Cy s
SH
418
SH
Cy s
SH
Acetyl CoA
CoA
734
O
734
734
Gly
Gly H
Gly
O
419
Cy s
SH
418
Cy s
S
O
419
Cy s
S
418
Cy s
SH
O
form ate
419
Cy s
S
418
Cy s
SH
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.
O
34
PFL: 1st Half of Reaction
Knappe Proposed Mechanism
734
Gly
734
734
Gly H
pyruvate
734
Gly H
Gly H
OH
419
Cy s
419
SH
Cy s S
OH
419
Cy s
O
418
419
Cy s S
Cy s S
O
Cy s
OH
O
418
Cy s
418
Cy s
SH
O
S
418
S
S
O
O
Acetyl CoA
CoA
734
419
Cy s
734
734
Gly
S
O
419
Cy s
734
Gly H
Gly H
S
419
Cy s
O
Gly H
S
O
419
Cy s
S
418
Cy s
S
O
H
418
Cy s
SH
418
Cy s
S
formate
418
Cy s
S
O
O
Knappe, J.; Elbert, S.; Frey, M.; Wagner, A.F.V. Biochem. Soc. Trans. 1993, 21, 731-734.
OH
O
35
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.
36
PFL: 1st Half of Reaction
Methacrylate Inhibition



Irreversible inhibition
14C labeled methacrylate confirmed consistent alkylation
of Cys418
Gly734• remains intact
CH2
O
O
O
O
pyruvate
O
methacrylate
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.
37
PFL: 1st Half of Reaction
Methacrylate Inhibition
734
734
Gly H
419
Cy s
418
Cy s
734
Gly H
SH
419
O
Cy s
418
Cy s
CH2
734
419
Cy s
S
H
S
CH2
O
S
O
418
CH2
Cy s
734
734
Gly H
O
SH
O
O
S
Gly H
Gly H
Gly H
734
Gly H
O
O
419
Cy s
SH
419
Cy s
O
419
SH O
Cy s
SH
Cy s
S
O
419
Cy s
S
418
Cy s
S
H
O
O
418
Cy s
O
S
O
418
Cy s
418
S
O
O
O
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.
38
PFL: 1st Half of Reaction
Currently Accepted Mechanism
734
734
Gly H
Gly
419
Cy s
SH
419
SH
418
pyruvate
734
734
Gly H
Cy s
S
419
Cy s
SH
418
Cy s
Gly H
SH
419
Cy s
O
SH O
O
O
418
Cy s
Cy s
S
O
418
Cy s
S
O
Acetyl CoA
CoA
734
734
734
Gly
734
Gly H
Gly H
Gly H
O
419
Cy s
418
Cy s
419
SH
S
Cy s
O
418
Cy s
419
Cy s
S
S
O
formate
418
Cy s
S
S
H
O
O
O
Frey, P.A.; Hegeman, A.D.; Reed, G.H. Chem. Rev. 2006, 106, 3302-3316.
419
Cy s
SH
O
418
Cy s
S
O
39
PFL: 2nd Half of Reaction
Polar Mechanism
Conventional acyl transfer by nucleophilic attack with radical
bystander

734
734
419
Cy s
419
Cy s
SH
Cy s
S
O
Gly
Gly
SH
S-CoA
418
734
734
Gly
Gly
419
Cy s
SH
418
Cy s
S
S-CoA
418
Cy s
S
O
S-CoA
419
Cy s
SH
418
Cy s
SH
O
Himo, F.; Eriksson, L.E. J. Am. Chem. Soc. 1998, 120, 11449-11455.
40
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
734
734
734
Gly
Gly H
Gly H
HS-CoA
419
Cy s
SH
Cy s
S
418
419
O
Cy s
S
Cy s
S
418
419
Cy s
SH
Cy s
S
HS-CoA
418
O
S-CoA
O
734
Gly H
419
Cy s
SH
S-CoA
734
734
Gly
734
Gly H
Gly H
418
Cy s
S
O
419
Cy s
SH
419
Cy s
S
418
Cy s
SH
418
Cy s
SH
AcS-CoA
419
Cy s
SH
418
Cy s
S
O
S-CoA
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.
41
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
42
Lysine 2,3-Aminomutase
(LAM)
+
H NH3
+
H3N
COO
H



H
LAM
H H
+
H3N
COO
H3N
+
H
First step in metabolism of lysine in Clostridia
Stereospecific reaction
Catalytic SAM and pyridoxal phosphate (PLP)
H
O
2
OH
O3PO
N
Chirpich, T.P.; Zappia, V.; Costilow, R.N.; Barker, H.A. J. Biol. Chem. 1970, 245, 1778-1789.
43
LAM:
Tritium Transfer Experiments
NH2
NH2
N
CH3 3H
3
H N
S+
O
CH3
S+
N
N
LAM
N
OH OH
OOC
NH+3
OH OH
+ NH3
+ NH
3
+H N
3
N
O
OOC
NH+3
N
N
COO
+H N
3
COO
3
H
3
H
+H N
3
COO
+
NH3
Baraniak, J.; Moss, M.L.; Frey, P.A. J. Biol. Chem. 1989, 264 1357-1360
44
LAM:
Proposed Mechanism
Ado
H
2
+H3NCH2CH2CH2 CH CH COO
OH
O3PO
-ly sine
ly sine
H
Ly s337 N
N
H
N
2
OH
O3PO
N
AdoH
Ado
+H3NCH2CH2CH2 CH CH COO
+H3NCH2CH2CH2 CH CH2 COO
H
N
2
OH
O3PO
H
N
2
N
OH
O3PO
N
AdoH
AdoH
+H3NCH2CH2CH2 CH CH COO
OH
O3PO
N
H
N
H
N
2
+H3NCH2CH2CH2 CH CH COO
OH
2
O3PO
N
Baraniak, J.; Moss, M.L.; Frey, P.A. J. Biol. Chem. 1989, 264 1357-1360
Danen, W.C.; West, C.T. J. Am. Chem. Soc. 1974, 96, 2447-2453.
45
LAM: Role of PLP
Chemical Model System
Br
CH3
H2C
C
H
N
CH3
CH3
COOEt
Bu3SnH, AIBN
H2C
C
H
N
COOEt
H3C
C
H
N
COOEt
B:C
1:13
B
A
65% yield
CH3
CH3
H2C
H
C
COOEt
N
H2C
C
N
H
H
COOEt
C
Han, O.; Frey, P.A. J. Am. Chem. Soc. 1990, 112, 8982-8983.
46
LAM:
Steady State Radical
EPR
+ NH3
+
H3N
COO
D
D D D
+ NH3
+
H3N
COO
D D
+H3NCH2CH2CH2 CH CH COO
D D
H
N
+NH3
+
H3N
2
D
OH
O3PO
COO
N
+ NH3
+
H3N
COO
13
C
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.
47
LAM: Analogue Radicals
4-Thia-L-lysine
+
H NH3
+
H3N
S
+
H3N
COO
S
H3N
+
+ NH
4
+
+
H3N
COO
H
SH
H
COO
+
O
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
48
LAM: Analogue Radicals
4-Thia-L-lysine
EPR
+ NH3
+
H3N
S
COO
+H3NCH2CH2S CH CH COO
+ NH3
S
H
N
COO
H3N
D D
2
OH
O3PO
N
+ NH3
+
H3N
S
COO
13
C
Wu, W.; Lieder, K.W.; Reed, G.H.; Frey, P.A. Biochemistry 1995, 34, 10532-10537.
49
LAM: Analogue Radicals
trans-4-Dehydrolysine
CH-PLP
H
+
H3N
CH-PLP
N
COO
H
+
H3N
COO
H H
Ado
N
H
AdoH
Wu, W.; Booker, S.; Lieder, K.W.; Bandarian, V.; Reed, G.H.; Frey, P.A. Biochemistry 2000, 39, 9561-9570.
50
LAM: Analogue Radicals
trans-4,5-Dehydrolysine
+ NH3
+
H3N
COO
+NH3
+
H3N
D
COO
D
EPR
A
B
+ NH3
+
H3N
COO
C
D
D
D
D
+ NH3
+
H3N
COO
D
D D
D + NH3
+
H3N
D
COO
D
D
D D
E
D D
Wu, W.; Booker, S.; Lieder, K.W.; Bandarian, V.; Reed, G.H.; Frey, P.A. Biochemistry 2000, 39, 9561-9570.
51
LAM: Currently
Accepted Mechanism
Ado
H
2
+H3NCH2CH2CH2 CH CH COO
OH
O3PO
-ly sine
ly sine
H
Ly s337 N
N
H
N
2
OH
O3PO
N
AdoH
Ado
+H3NCH2CH2CH2 CH CH COO
+H3NCH2CH2CH2 CH CH2 COO
H
N
2
OH
O3PO
H
N
2
N
OH
O3PO
N
AdoH
AdoH
+H3NCH2CH2CH2 CH CH COO
OH
O3PO
N
H
N
H
N
2
+H3NCH2CH2CH2 CH CH COO
OH
2
O3PO
N
Frey, P.A.; Hegeman, A.D.; Reed, G.H. Chem. Rev. 2006, 106, 3302-3316.
52
LAM: Enzyme Control
ENDOR 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.
53
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
54
Spore Photoproduct-lyase
(SPP lyase)
O
O
SPP ly ase
NH
HN
N
N
R
R
O
O
O
O
UV irradiation
HN
O
NH
+
N
N
R
R
O
Spore Photoproduct



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.
55
Friedel, M.G.; Berteau, O.; Pieck, J.C.; Atta, M.; Ollagnier-de-Choudens, S.; Fontecave, M.; Carell, T. Chem. Commun., 2006, 445-447.
SPP lyase:
Chemical Model System
O
Bu3SnH, (Bu3Sn)2
AIBN, PhH
N
N
N
N
N
N
(6)
O
O
O
O
O
(6)
SPh
N
O
O
N
N
O
N
O
85%

C6 radical of spore photoproduct can undergo β-scission
Mehl, R.A.; Begley, T.P. Org. Lett. 1999, 1, 1065-1066.
56
SPP lyase:
Tritium Labeling Experiments
SAM
O
O
NH
HN
(6)
N
O
3
3
R
O
HN
O
H
H
C3H3
C
3
H2
N
O
R
O
NH
N
N
R
R
O
 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
57
SPP lyase:
Proposed Mechanism
Guo, J.D.; Luo, Y.; Himo, F. J. Phys. Chem. B 2003, 107, 11188-11192.
58
Radical SAM Enzymes:
Conclusions

Large family of over 600 postulated enzymes



Shared mechanism for formation of adenosyl radical
Independent and unique uses of adenosyl radical



< 5% characterized
Generate protein or substrate radical
Diverse reactions
Many unique and powerful mechanisms yet to discover

Novel radical chemistry
59
Acknowledgments

Hans Reich


Perry Frey
Ieva Reich
Practice Talk Attendees






Melissa Boersma
Seth Horne
Luke Lavis
Amanda King
Reich Group


Kris Kolonko
Amanda Jones






Erin McElroy
Katie Partridge
Kim Peterson
Kathy Van Heuvelen
Michael
Mason
60
EXTRA SLIDES
61
PFL:
Chemical Model Support
O
H2O2
HO-O OH
FeSO4
O OH
O
+
COOEt

COOEt
COOEt
COOEt
OH
Minisci et al. reported cleavage of α-keto esters with Fenton’s
reagent
62
PFL:
Mercaptopyruvate Inhibitor
63
PFL
Hypophosphite inhibitor
64
LAM: Analogue Radicals
trans-4,5-Dehydrolysine
[4Fe-4S]+1
Equiv of [4Fe-4S]+
Equiv of Organic Radical
kloss = 2.6 ± 0.4 min-1
CH-PLP
H
+
H 3N
N
COO
H
kform = 2.9 ± 0.6 min-1
Time (min)
Wu, W.; Booker, S.; Lieder, K.W.; Bandarian, V.; Reed, G.H.; Frey, P.A. Biochemistry 2000, 39, 9561-9570.
65
Reduction Potentials
PFL-AE
[4Fe-4S]+
V
-0.2
-0.4
-0.6
Nonenzymatic
trialkyl
sulfonium
Estimated SAM
-0.8
-1.0
-1.2
-1.4
-1.6
-1.8
0.27 V
Keq≈ 10-5
LAM [4Fe-4S]+
LAM [4Fe-4S]+
with SAM
with SAM and
lysine
ln K = nE°/ 0.0257 at 25° C
E° = 0.27 V
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.
66
Acetyl Coenzyme A
67
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
sample
γ-ray emitter
detector
Drago, R.S. Physical Methods for Chemists; Sauders College:Orlando, FL, 1977; 2nd Ed, pp 626-645.
68
Solomon, E.I.; Lever, A.B.P., Eds.; Inorganic Electronic Structure and Spectrocopy; Wiley-Interscience: New York, NY, 1999; Vol. 1, pp 161-211.
Identification of Fe-S cluster
in PFL-AE

Mössbauer Spectroscopy
2+
1+
S
S
Fe Fe
Fe
S
S
S
Fe
Fe
1+
S
S
Fe
Fe
S
Fe
S
S
Fe
Na2S2O4
1+/2+
S
Fe
Fe
S
Fe
S



S
Fe
[4Fe-4S] usually stabilized by 4 Cys
Site-directed mutagenesis of CxxxCxxC
Labile Fe-S cluster with site-differentiated cluster

Precedent in aconitase
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.
69
A Unique Iron Site
without SAM
with SAM
difference spectrum
Krebs, C.; Broderick, W.E.; Henshaw, T.F.; Broderick, J.B.; Huynh, B.H. J. Am. Chem. Soc. 2002, 124, 912-913.
70
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.
71
Methionine Coordination:
Se EXAFS
Se-Methionine
NH2
N
CH3
+
Se
Se-C
N
N
N
O
Se-Fe
LAM
OOC
NH+3
OH OH
PFL-AE
Se
BioB
OOC
NH+3
72
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.
SAM Coordination:
ENDOR Spectroscopy

PFL-AE


Isotropic coupling indicated local orbital overlap
Assigned to a dative interaction between sulfonium and
sulfide
+
73
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.
Mechanistic Differences
Catalytic Enzyme:
LAM
Stoichiometric Enzyme:
PFL and BioB
74
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
EPR Spectroscopy
MS
1/2
-1/2
Energy
1/2
MI
-1/2
1/2

Hyperfine Splitting
 By neighboring
nucleus with nuclear
spin
 Magnitude of splitting
depends on nucleus
-1/2
H
D
Magnetic Field
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.
75