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Radical Enzymes
PPO
O
HO
N
PPO
O
OH
HO
Jian Jin
MacMillan Group Meeting
November 23, 2015
N
Enzymes
■ Definition
■ Enzymes are macromolecular biological catalysts.
■ Catalyze more than 5,000 biochemical reaction types.
■ Most enzymes are proteins, a few are catalytic RNA molecules.
■ Structure
■ Enzymes are linear chains of amino acids that fold to produce a three-dimensional structure.
■ The sequence of the amino acids specifies the structure.
■ The structure determines the catalytic activity of the enzyme.
Blake, C. C.; Koenig, D. F.; Mair, G. A.; North, A. C.; Phillips, D. C.; Sarma, V. R. Nature, 1965, 206, 757.
Enzymes
■ Substrate Binding
Enzymes must bind their substrates before they can catalyze any chemical reaction.
■ "Key and lock" model: In 1894, Emil Fischer proposed: the enzyme and the substrate fit exactly into one another.
■ Induced fit model: In 1958, Daniel Koshland suggested a modification: the active site is continuously reshaped
by interactions with the substrate.
no exact fit of
substrate and enzyme
substrate binding
enzyme changes shape
by induced fit
Fischer, E. Berichte der Deutschen Chemischen Gesellschaft zu Berlin, 1894, 27, 2985.
Koshland, D. E. PNAS, USA, 1958, 44, 98.
Enzymes
■ Cofactors
■ Some enzymes do not need additional components to show full activity. Others require non-protein molecules called
cofactors to be bound for activity.
■ Cofactors can be either inorganic or organic compounds.
S-cys
S
cys-S
Me
Fe
Me
Fe
S
cys-S Fe
N
Fe 4S4 cluster
S
N
FeII
S
S-cys
O
HN
O
N
N
Fe
N
Me
N
Me
Me
N
Me
heme B
HO 2C
NH 2
OH
HO
N
CO2H
N
OH
N
O
O
HO
O
P
O
O
P
NH 2
N
O
O
NH 2
OH
OH
N
OH
N
O
O
flavin adenine dinucleotide (FAD)
left half of FAD
flavin mononucleotide (FMN)
N
O
HO
O
P
O
O
P
N
O
OH
OH
OH
N
OH
OH
nicotinamide adenine dinucleotide (NAD)
Radical Enzymes
■ Definition
The term radical enzyme describes those enzymes that catalyze reactions in which radicals participate.
■ Radicals of Biology
O
S
O
OH
O
O
OH
NH 2
O
cysteine thiyl radical
(cysteinyl)
H 2N
NH 2
tryptophan cation radical
Me
Me
Me
N
N
O
N
N
FeIV
N
N
Me
O
S
OH
O
N
FeIV
N
N
Me
HO 2C
OH
NH 2
HN
Me
NH 2
N
OH
glycine carbon radical
(glycyl)
tyrosine oxy radical
(tyrosyl)
coenzyme B12 / SAM
N
OH
CO2H
Me
HO 2C
S
N
Me
CO2H
CYP450 compound I
5'-deoxyadenosyl radical
Buckel, W.; Golding B. T. Radical Enzymes in Encyclopedia of Radicals in Chemistry, Biology and Materials, 2012, John Wiley & Sons.
Cytochromes P450
■ Introduction
The most common reaction catalyzed by cytochromes P450 is a monooxygenase reaction:
RH + O2 + NADPH + H + → ROH + H 2O + NADP +.
■ Oxygen Rebound Mechanism in P450-catalyzed C−H Hydroxylation
O2, 2 H +, 2 e−
Me
H 2O
Me
Me
Me
N
N
N
FeIII
N
Me
HO 2C
S
O
N
FeIV
N
N
S
N
Me
Me
Me
HO 2C
CO2H
resting ferric enzyme
ROH
CO2H
R−H
compound I
Me
R
R Me
N
OH N
FeIV
N
Me
S
N
Me
HO 2C
CO2H
rebound intermediate
Liu, W.; Groves, J. T. Acc. Chem. Res. 2015, 48, 1727.
Ribonucleotide Reductases
■ Introdution
Ribonucleotide reductases (RNR) are enzymes that catalyze the formation of deoxyribonucleotides from
ribonucleotides. Deoxyribonucleotides in turn are used in the synthesis of DNA.
■ Classes of RNRs
■ Class I enzymes: aerobic, di-iron-oxo (class Ia) or dimanganese-oxo (class Ib) or Fe(III)-(O) 2-Mn(IV) (class Ic).
■ Class II enzymes: anaerobic but can survive when exposed to O2, coenzyme B12.
■ Class III enzymes: strictly anaerobic, SAM.
pathway for proton-coupled electron transfer (top)
NH 2
PPO
O
ribonucleotide
H
N
O
H
HO
HS
OH
NH 2
OH
residue
O
OH
residue
O
MnII
O
Tyr-OH
H 2O
O
MnII
O
MnII2-NrdF
Nrdl
HO 2−
MnIII
O
residue
O
NH 2
OH
Tyr-OH
OH
Tyr-OH
H 2O
O
MnIII
O
OH
Nrdl?
O
MnIII
MnIV
O
O
O
HO 2−, e− (FMNH 2)
Tyr-O•
H 2O
O
MnIII
MnIII
O
O
O
MnIII2 /Tyr•-NrdF
tyrosyl radical generated by dimanganese-oxo species (bottom)
Sjoberg, B.-M. Science, 2010, 329, 1475.
Ribonucleotide Reductases
■ Mechanism for RNR Catalyzed Reduction of NDPs (E. coli)
C439
C439
C439
S
SH
SH
PPO
O
E441
H
O
PPO
N
O
H
HO
OH
O
NH 2
N437
OH
H
O
E441
OH
O
H
NH 2
N437
SH S
O
pK a ~ 8
C462 C225
NH 2
N437
C439
C439
S
SH
SH
PPO
H
O
H
HO
H
O
H
HO
E441
O
PPO
N
O
H
N437
NH 2
S
S
C462 C225
N
H
O
O
E441
O
O
SH S
C462 C225
C439
N
N
O
O
C462 C225
O
O
HO
E441
SH SH
PPO
E441
O
H
O
glumate
O
PPO
N
H
OH
O
N437
NH 2
S
S
C462 C225
O
N437
NH 2
S
S
C462 C225
Zipse, H. et al. J. Am. Chem. Soc. 2009, 131 , 200.
Coenzyme B12 -Dependent Enzymes
■ Introduction
■ The Co-C bond is reltively weak (BDE ~ 31.2 KCal/mol), however the coenzyme B12 is rather stable in water at
30 °C (τ1/2 1.9 years).
■ The binding to the enzyme partner and the arrival of a substrate molecule initiates cleavage of the Co-C bond.
■ Coenzyme B12 operates with two types of enzymes: the eliminases and the mutases.
CONH2
CONH2
Me
Me
Me
AdoCH2-Cbl
CONH2
H 2NOC
N
Me
R
N
Co
H
N
H 2NOC
N
NH
AdoCH2• + •Cbl
Me
Me
CONH2
Me Me
O
N
Me
N
Me
HO
O
Me
O
P
O
PH
SH
O
AdoCH3 + •Cbl
S•
P•
Minimal mechanism for coenzyme B12 -dependent enzymatic reactions
coenzyme B12 (R = 5'-deoxyadenosyl)
Brown, K. L. Chem. Rev. 2005, 105 , 2075.
Coenzyme B12 -Dependent Enzymes
■ Eliminases
H
R1
OH
OH
R2
R1
OH/NH2
O
R2
R1
OH/NH2
■ Glycerol dehydratase
R2
+
H 2O/NH3
OH
HO
OH
O
HO
H
OH
■ Propane-1,2-diol dehydratase
O
OH
Me
Me
H
■ Ribonucleotide reductases
PPO
O
N
PPO
NADPH +
H+
N
O
H
HO
■ Ethanolamine ammonia lyase
OH
HO
OH
H 2N
Me
O
H
Buckel, W.; Golding B. T. Radical Enzymes in Encyclopedia of Radicals in Chemistry, Biology and Materials, 2012, John Wiley & Sons.
Coenzyme B12 -Dependent Enzymes
■ Carbon-skeleton Mutases
H
C
R1
R1
R1
C
C
■ Glutamate mutase
O2C
O2C
H
C
H
NH 3
R1
O2C
O2C
H
NH 3
■ 2-Methylene-glutarate mutase
O2C
H
O2C
H
O2C
H
O2C
H
O2C
■ Methylmalonyl-CoA mutase
O
O2C
H
SCoA
O2C
H
O
O2C
H
SCoA
O
Me
H
H
SCoA
Me
H
CO2
H
NH 3
CH3
O2C
H
■ Isobutyryl-CoA mutase
O2C
H
H 3C
H
H
H
H 3C
Me
H
O
SCoA
H 3C
O
SCoA
O
SCoA
Buckel, W.; Golding B. T. Radical Enzymes in Encyclopedia of Radicals in Chemistry, Biology and Materials, 2012, John Wiley & Sons.
Coenzyme B12 -Dependent Enzymes
■ Amino Mutases
H
N
R1
R1
R1
N
N
N
H
R1
■ β-Lysine-5,6-aminomutase
NH 2
NH 3
H 2N
6
CO2
5
NH 3
CO2
H 3C 5
6
■ Ornithine-4,5-aminomutase
4
CO2
H 2N 5
H 3C
5
NH 3
4
NH 2
CO2
NH 3
O
HO
HO
condensation
HAT
O
Me
OH
P
O
hydrolyzation
HAT
N
coenzyme pyridoxal phosphate
4
N 5
Ar
NH 3
4
5
CO2
N
Ar
CO2
4
5
N
NH 3
azacyclopropylcarbinyl radical
CO2
NH 3
Ar
Buckel, W.; Golding B. T. Radical Enzymes in Encyclopedia of Radicals in Chemistry, Biology and Materials, 2012, John Wiley & Sons.
SAM radical Enzymes
■ Introduction
To date, there are about 40 different radical reactions catalyzed by characterized SAM radical enzymes.
The 5'-deoxyadenosyl radical initiates catalysis generally by hydrogen atom abstraction from the substrate.
■ Classes of SAM radical enzymes
■ Recylcling SAM radical enzymes: the resulting 5'-deoxyadenosine is recycled to regenerate SAM.
■ Irreversible SAM radical enzymes: 5'-deoxyadenosine is dumped irreversibly.
NH 2
N
N
N
N
Me
O
5'-deoxyadenosine
S
OH
OH
H
N
S
cys-S
ferredoxin or flavodoxin e
( E' = −0.5 V)
Fe
cys-S
HS
Fe
O
S
Fe
methionine
S
Fe
S-cys
O
Fe 4S4 cluster
Mode of SAM (S-adenosyl methionine) binding to the fourth iron of the cluster
Roach, P. L. Curr. Opin. Chem. Biol. 2011, 15 , 267.
SAM radical Enzymes
■ Recycling SAM Radical Enzymes
■ Lysine-2,3-aminomutase
In 1989, Perry A. Frey discovered hydrogen transfer from the 5'-methylene group of SAM to α- and βlysine, which means 5-deoxyadenosyl radical is involved and could be regenerated.
NH 3
H 2N
6
lysine-2,3-aminomutase
CO2
5
H 2N
6
SAM
NH 3
(S)-α-lysine
(S)-β-lysine
O
HO
HO
O
Me
CO2
5
β-lysine-5,6-aminomutase
OH
P
coenzyme B12
O
NH 2
N
coenzyme pyridoxal phosphate
NH 3
CO2
H 3C 5
6
■ DesII
H
H
Me
H 2N
HO
H
H
H O
DesII
H
OH
OTDP
SAM
Me
H
H
O
H O
H
OH
OTDP
D-glucose derivative
Buckel, W.; Martins, B. M.; Messerschmidt, A.; Golding, B. T. Biol. Chem. 2005, 386, 951.
SAM radical Enzymes
■ Recycling SAM Radical Enzymes
■ Spore Photoproduct Lyase, recycling or irreversible? Recent experiments suggest No.
Spore photoproduct lyase has to repair DNA lesions that occasionally occur in bacteria germinated
from spores.
O
HN
O
Me
NH
5
O
N
O
6
N
HN
O
O
Ado-CH2•
spore photoproduct
HN
O
N
+ e− + H +
NH
5
NH
5
N
N
O
O
Me Me
N
O
O
O
Ado-CH3
UV-B
O
Me
HN
O
O
Me
NH
5
N
N
O
two adjacent thymidines
Chandor-Proust, A.; Berteau, O.; douki, T. et al. J. Biol. Chem. 2008, 283, 36361.
SAM radical Enzymes
■ 7 Known Irreversible SAM-Glycine-Cysteine Radical Enzymes
■ RNR III (refer to the RNR part)
■ Pyruvate formate lyase
O
cys-S•
HO
Me
O
O
HO
O
HO
cys-SH
+
Me
S-cys
O
cys-S
Me
O
HO
H
O
+
CoASH
O
CoA-S
Me
formate
pyruvate
■ 2-Oxobutyrate formate lyase (similar to the above)
■ 2-(1-methylpentyl)succinate synthase
Me
cys-S•
Me
Me
Me
O2C
CO2
CO2
CO2
CO2
fumarate
Me
Me
cys-SH
CO2
Me
Me
C-2: Δ BDE ~ 9.6 Kcal/mol
C-1: Δ BDE ~ 12 Kcal/mol
■ Benzylsuccinate synthase (similar to the above)
Buckel, W.; Golding B. T. Radical Enzymes in Encyclopedia of Radicals in Chemistry, Biology and Materials, 2012, John Wiley & Sons.
SAM radical Enzymes
■ 7 Known Irreversible SAM-Glycine-Cysteine Radical Enzymes
■ Glycerol dehydratase (similar to the version of coenzyme B12 -dependent enzyme)
■ p-Hydroxyphenylacetate decarboxylase
O
O
O
HO
O
HO
p-hydroxyphenylacetate
cys-S•
cys-S−
Kolbe-type
CO2 + H +
CH3
O
HO
p-cresol
2 H+
p-cresol radical anion
Buckel, W.; Golding B. T. Radical Enzymes in Encyclopedia of Radicals in Chemistry, Biology and Materials, 2012, John Wiley & Sons.
SAM radical Enzymes
■ Irreversible SAM Radical Enzymes: PylB
H 2N
4
CO2
H 2N
4
H 2N
CO2
CO2
H 2N
NH 3
NH 3
NH 3
Me
CO2
NH 3
(2R, 3R)-3-methylornithine
(S)-α-lysine
■ Irreversible SAM Radical Enzymes: HydG
O
O
O
HO
NH 3
(S)-tyrosine
Ado-CH2•
O
-
H+
+
O
O
NH 3
O
O
NH 2
dehydroglycine
Gaston, M. A.; Zhang, L.; Green-Church, K. B.; Krzycki, J. A. Nature, 2011, 471, 647.
SAM radical Enzymes
■ Irreversible SAM Radical Enzymes: Sulfur insertion
The abstraction of a hydrogen atom bound to carbon is followed by insertion of sulfur derived from a second iron-sulfur
cluster of SAM radical enzyme.
SubH
Ado-CH2•
Fe 2S2 cluster
Sub
Sub−S
Pdt
■ Examples of sulfur insertion by SAM radical enzymes
O
Me
O
HN
NH
H
Protein
H
S
S
S
D-biotin
HO
protein-bound lipoic acid
O
Protein
O
CO2H
S
HN
Protein
thiomethylated aspartate
in the ribosomal protein S12
Atta, M.; Mulliez, E.; Arragain, S. et al. Curr. Opin. Struct. Biol. 2010, 20, 684.
Reactions Involving Ketyl Radicals
■ Backgroup of CoASH and ATP
■ Coenzyme A (CoASH): react with carboxylic acids to form thioesters, followed by a variety of enzyme-catalyzed
transformation.
■ Adenosine triphosphate (ATP): a nucleoside triphosphate used in cells, a coenzyme often called the "molecular
unit of currency" of intracellular energy transfer.
NH 2
N
O
coenzyme A (CoASH)
HS
O Me
N
H
Me
O
N
H
OH
O
P
O
O
O
P
N
O
N
N
O
O
O
O
P
OH
O
O
NH 2
N
O
O
P
O
O
O
P
O
O
O
P
N
O
N
N
adenosine triphosphate (ATP)
O
O
OH
OH
Seebach, D. Angew. Chem. Int. Ed. 1979, 18 , 239.
Buckel, W.; Keese, R. Angew. Chem. Int. Ed. 1995, 34, 1502.
Buckel, W.; Golding, B. T. Chem. Soc. Rev. 1996, 25, 329.
Reactions Involving Ketyl Radicals
■ 2-Hydroxyacyl-CoA dehydratase
■ SET causes the acidity of β-proton to increase by 26 orders of magnitude.
■ Use of just one electron provides an explanation as to how an "impossible" dehydration can occur.
H pK a ~ 40
O
CoAS
O
− H 2O
CoAS
R
R
OH
(E)-2-enoyl-CoA
(R)-2-hydroxyacyl-CoA
e−
2 ADP + 2 P i
E red ~ −0.9 V
2 ATP + 2 H 2O
ferredoxin−
O
H+
H
CoAS
R
H+
HOH
O
CoAS
O
H pK a ~ 14
R
CoAS
R
OH
ketyl radical
enoxy radical
allylic ketyl radical
Smith, D. M.; Buckel, W.; Zipse, H. Angew. Chem. Int. Ed. 2003, 42, 1867.
Reactions Involving Ketyl Radicals
■ 4-Hydroxybutyryl-CoA dehydratase
O
H pK a ~ 40
O
− H 2O
OH
CoAS
CoAS
H pK a ~ 8
O
4-hydroxybutyryl-CoA
crotonyl-CoA
N
HN
Me
H+
H+
O
O
N
N
Me
HO
H
OH
CoAS
Me
O
R
CoAS
flavodoxin+: FAD
enolate
dienolate
flavodoxin+
ferredoxin−
e−
O
CoAS
H+
H pK a ~ 14
OH
enoxy radical
HOH
O
O
CoAS
H+
OH
allylic ketyl radical
CoAS
dienoxy radical
Buckel, W.; Golding, B. T. Annu. Rev. Microbiol. 2006, 60, 27.
Reactions Involving Ketyl Radicals
■ Acyl-CoA Dehydrogenase
O
H
O
CoAS
H
O
O
Me
CoAS
H pK a ~ 8
Me
CoAS
H+
CoAS
Me
e−
e− + H +
butyryl-CoA
Me
ketyl radical
enolate
crotonyl-CoA
H+
FAD
FADH•
FADH 2
■ Chorismate Synthase
O
O
O
H
O
O
P
O
O
− HPO 42−
FMNH -
CO2
O
OH
O
O
OH
+ eO
− e-
O
O
O
P
O
O
O
OH
O
O
pKa ~ 14
− PO 43−
H
O
CO2
H
CO2
O
OH
O
− H+
O
CO2
CO2
OH
Ghisla, S.; Thorp, C. Eur. J. Biochem. 2004, 271, 494.
Kitzing, K.; Auweter, S.; Amrhein, N.; Macheroux, P. J. Biol. Chem. 2004, 279, 9451.
Reactions Involving Ketyl Radicals
■ DNA photolyase
O
O
HN
O
Me Me
O
N
O
N
O
HN
+ e−
NH
5
6
Me Me
NH
N
N
O
O
thymidine dimer
FADH −*
350-450 nm UV-A
290-320 nm UV-B
methylenetetrahydrogolate
λmax = 380 nm
FADH −
O
O
N
O
O
Me Me
HN
FADH•
N
Me Me
HN
NH
O
+ e−
O
O
N
NH
N
O
two adjacent thymidines
Mees, A.; Klar, T.; Gnau, P. et al. Science, 2004, 306, 1789.
FADH 2-dependent Hydratase
■ 2-Haloacrylate hydratase
The hydratase catalyzes the hydration of 2-chlroacrylate to pyruvate and chloride, while a simple hydration
of chloroacrylate would lead to 2-chloro-3-hydroxypropionate.
+ e−
Cl
Cl
CO2
2-chlroacrylate
H 2C
FADH −
+ H+
FADH•
Cl
Cl
H 3C
CO2
CO2
+ e−
H 3C
CO2
+ OH−
Cl
HO
Cl
CO2
2-chloro-3-hydroxypropionate
H 3C
O + H + + Cl−
OH
CO2
H 3C
CO2
pyruvate
Mowafy, A. M.; Kurihara, T.; Kurata, A. et al. Appl. Environ. Microbiol. 2010, 76, 6032.
Reaction of Aromatic Compounds
■ Class I BCR
2 ADP + 2 P i
2Fdox
2 ATP + 2 H 2O
2Fdred
O
O
+
SCoA
H
SCoA
H
2 H+
e−
H
H
+ H+
O
O
O
+
+ H+
SCoA
e−
SCoA
H
H
H
SCoA
−
H
■ Protochlorophllide reductase
Me
N
Me
Me
N
N
NADPH +
MgII
N
H+
+ hυ
Me
Me
HO 2C
N
MgII
2 Fd red + 2 H + + 2 ATP
N
Me
N
N
Me
Me
HO 2C
MeO 2C
O
MeO 2C
O
Boll, M. BioChim. Biophys. Acta. 2005, 1707 , 34.
Rees, D. C.; Howard, J. B. Curr. Opin. Chem. Biol. 2000, 4, 559.
Isoprenoid Biosynthesis
■ (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE) and reductase (LytB)
Cleavage of the cyclodiphosphate ring to a tertiary carbocation is followed by one-electron reduction to give
a β-hydroxyalkyl radical that eliminates the C-3 hydroxyl group in the manner of RNR or diol dehydratase.
O
Me
O
2
OH
O
P
3
O O
P O
Me
GcpE
3
O
OH
Me
GcpE
3
OPP
OH
OH
OH
[Fe 4S 4]+
OH
[Fe 4S 4]2+
''E'' +
GcpE
Me
LytB
Me
OPP
+ 2 e−
− "E"−OH
OPP
− "E"−OH
Me
GcpE
OPP
OPP
OH
OH
[Fe 4S 4]2+
''E'' +
[Fe 4S 4]+
LytB H +
Me
Me
IPP
OPP
+
DMAPP
Me
OPP
''E'' +: electrophile
Tritsch, D.; Hemmerlin, A.; Bach, T. J.; Rohmer, M. FEBS Lett. 2010, 584, 129.
Biotechnological Application
■ Coenzyme B12 -dependent glycerol dehydratase
■ The only one radical enzyme currently applied in biotechnology.
■ The product 1,3-propanediol, a valuable monomer for polyesters, from which fabrics are made.
■ The company Dupont advertises the fabrics as "Clothes from Corn".
OH
HO
OH
glycerol dehydratase
HO
O
reductase
HO
OH
H
glycerol
3-hydroxypropanal
1,3-propanediol
Toraya, T. Cell. Mol. Life Sci. 2000, 57, 106.