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Radical Enzymes
Jian JinMacMillan Group Meeting
November 23, 2015
NO
OHHO
PPON
O
HO
PPO
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 BindingEnzymes 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.
Fischer, E. Berichte der Deutschen Chemischen Gesellschaft zu Berlin, 1894, 27, 2985.Koshland, D. E. PNAS, USA, 1958, 44, 98.
no exact fit ofsubstrate and enzyme
substrate binding
enzyme changes shapeby induced fit
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.
Fe
S
S
S
FeFe
Fe
SFe4S4 cluster
S-cys
S-cys
cys-S
cys-S
N N
NN
Me
Me
Me Me
HO2C CO2Hheme B
FeII
flavin mononucleotide (FMN)
flavin adenine dinucleotide (FAD)
nicotinamide adenine dinucleotide (NAD)
N
N
HN
NO
OMe
Me
O
OH
OHHO
PP
HO O OH
O
N
NN
N
NH2
O
OHOH
O
left half of FAD
OP
P
HO
N
NN
N
NH2
O
OHOH
O
O
OHOH
N
NH2
OO
O
OO
OH
Radical Enzymes
■ DefinitionThe term radical enzyme describes those enzymes that catalyze reactions in which radicals participate.
■ Radicals of Biology
Buckel, W.; Golding B. T. Radical Enzymes in Encyclopedia of Radicals in Chemistry, Biology and Materials, 2012, John Wiley & Sons.
NH2
S
O
OH
cysteine thiyl radical(cysteinyl)
NH2O
O
OH
tyrosine oxy radical(tyrosyl)
H2NO
OH
glycine carbon radical(glycyl)
N N
NN
Me
Me
Me Me
HO2C CO2H
CYP450 compound I
FeIV
O
S
N
NN
N
NH2
O
OHOH
5'-deoxyadenosyl radical
N N
NN
Me
Me
Me Me
HO2C CO2H
FeIV
S
O
coenzyme B12 / SAM
tryptophan cation radical
NH2
O
OH
HN
Cytochromes P450
■ IntroductionThe most common reaction catalyzed by cytochromes P450 is a monooxygenase reaction: RH + O2 + NADPH + H+ → ROH + H2O + NADP+.
■ Oxygen Rebound Mechanism in P450-catalyzed C−H Hydroxylation
Liu, W.; Groves, J. T. Acc. Chem. Res. 2015, 48, 1727.
N N
NN
Me
Me
Me Me
HO2C CO2H
compound I
FeIV
O
SN N
NN
Me
Me
Me Me
HO2C CO2H
FeIII
S
N N
NN
Me
Me
Me Me
HO2C CO2H
FeIV
S
OH
rebound intermediate
resting ferric enzyme
O2, 2 H+, 2 e− H2O
R−H
RR
ROH
Ribonucleotide Reductases
■ IntrodutionRibonucleotide reductases (RNR) are enzymes that catalyze the formation of deoxyribonucleotides fromribonucleotides. Deoxyribonucleotides in turn are used in the synthesis of DNA.
■ Classes of RNRs
Sjoberg, B.-M. Science, 2010, 329, 1475.
■ 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.
MnIIO O
MnII
O O
MnII2-NrdF
Tyr-OH
MnIIIO
MnIII
O O
H2O
HO2−
Nrdl
Tyr-OH
HO2−, e− (FMNH2)
Nrdl?MnIII
OMnIV
O O
H2OTyr-OH
OMnIII
OMnIII
O O
H2OTyr-O•
O
MnIII2/Tyr•-NrdF
OH
O
OHNH2
OH
O
OHNH2
NH2
HS
O
OH
O
OHHO
HH
PPO
pathway for proton-coupled electron transfer (top)
residue residue residueribonucleotide
tyrosyl radical generated by dimanganese-oxo species (bottom)
N
Ribonucleotide Reductases
■ Mechanism for RNR Catalyzed Reduction of NDPs (E. coli)
Zipse, H. et al. J. Am. Chem. Soc. 2009, 131 , 200.
NO
OHHO
HH
PPO
NH2OSH SH
O
O
S
C439
E441
N437 C462 C225
NO
OHHO
H
PPO
NH2OSH S
O
O
SH
C439
E441
N437 C462 C225
NO
O
H
PPO
NH2OSH S
O
OH
SH
C439
E441
N437 C462 C225
NO
HHO
HH
PPO
NH2OS S
O
O
S
C439
E441
N437 C462 C225
NO
HHO
H
PPO
NH2OS S
O
O
SH
C439
E441
N437 C462 C225
NO
HO
H
PPO
NH2OS S
O
OH
SH
C439
E441
N437 C462 C225
HpKa ~ 8
glumate
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.
Brown, K. L. Chem. Rev. 2005, 105 , 2075.
Minimal mechanism for coenzyme B12-dependent enzymatic reactions
AdoCH2-Cbl
AdoCH2• + •CblSH
S• P•
PH
AdoCH3 + •Cbl
coenzyme B12 (R = 5'-deoxyadenosyl)
N N
NN
MeMe
Me
Co
MeCONH2
CONH2
H2NOCCONH2
Me
H
H2NOC
Me
CONH2MeMe
NHO
OMe P
OO O
HO
N
N
Me
Me
R
Coenzyme B12-Dependent Enzymes
■ Eliminases
Buckel, W.; Golding B. T. Radical Enzymes in Encyclopedia of Radicals in Chemistry, Biology and Materials, 2012, John Wiley & Sons.
R1R2
H OH
OH/NH2
R1R2
OH
OH/NH2R1
R2
O+ H2O/NH3
HO OH■ Glycerol dehydrataseOH
HO O
MeOH■ Propane-1,2-diol dehydratase
OH
MeO
H2NOH
■ Ribonucleotide reductases
Me O
NO
OHHO
PPO NO
HO
PPONADPH + H+
■ Ethanolamine ammonia lyase
H
H
H
H
Coenzyme B12-Dependent Enzymes
■ Carbon-skeleton Mutases
Buckel, W.; Golding B. T. Radical Enzymes in Encyclopedia of Radicals in Chemistry, Biology and Materials, 2012, John Wiley & Sons.
R1
C
R1
C R1
C
■ Glutamate mutase
■ 2-Methylene-glutarate mutase
■ Methylmalonyl-CoA mutase
■ Isobutyryl-CoA mutase
H
R1
CH
H
O2CH
O2C HNH3
H3C
O2CH
CO2
NH3H
HHO2C
O2C CH3HO2C
O2C
SCoAO
O2CH
H
OO2CH
H3C SCoA
SCoAO
MeH
H
OMeH
H3C SCoA
H
O2CO2C H
NH3
HO2C
O2CH
O2C
O
SCoA
MeH
O
SCoA
Coenzyme B12-Dependent Enzymes
■ Amino Mutases
Buckel, W.; Golding B. T. Radical Enzymes in Encyclopedia of Radicals in Chemistry, Biology and Materials, 2012, John Wiley & Sons.
R1
N
R1
N R1
N
■ β-Lysine-5,6-aminomutase
■ Ornithine-4,5-aminomutase
H
R1
NH
H2N CO2
NH3
6 5 H3CCO2
NH3
65
NH2
H2NCO2
5NH3
4 H3C CO25
NH3
4
NH2
NMe
HO
O
OP
O
OHHO
coenzyme pyridoxal phosphate
CO25
NH34
Ar
NCO2
5NH3
4
Arazacyclopropylcarbinyl radical
NCO25
NH3
4
N
Ar
condensationHAT
hydrolyzationHAT
SAM radical Enzymes
■ IntroductionTo 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.
Roach, P. L. Curr. Opin. Chem. Biol. 2011, 15 , 267.
Fe
HS
S
S
FeFe
Fe
S
Mode of SAM (S-adenosyl methionine) binding to the fourth iron of the cluster
S-cys
cys-S
cys-S
HN
O
S
Me
N
N N
N
NH2
O
OH OH
5'-deoxyadenosine
methionine
Fe4S4 cluster
ferredoxin or flavodoxin( E' = −0.5 V) e
■ 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.
O
SAM radical Enzymes
■ Recycling SAM Radical Enzymes
Buckel, W.; Martins, B. M.; Messerschmidt, A.; Golding, B. T. Biol. Chem. 2005, 386, 951.
H2N CO2
NH3
6 5
H3CCO2
NH3
65
NH2
H2N CO2
6 5NH3
(S)-α-lysine (S)-β-lysine
■ Lysine-2,3-aminomutase
lysine-2,3-aminomutase
β-lysine-5,6-aminomutase
NMe
HO
O
OP
O
OHHO
coenzyme pyridoxal phosphate
SAM
coenzyme B12
OH
H2N
H
HO
H
OTDPOHH
Me
H
D-glucose derivative
OH
OTDPOHH
Me
H
O
H
HDesII
SAM
■ DesII
In 1989, Perry A. Frey discovered hydrogen transfer from the 5'-methylene group of SAM to α- and β-lysine, which means 5-deoxyadenosyl radical is involved and could be regenerated.
SAM radical Enzymes
■ Recycling SAM Radical Enzymes
Chandor-Proust, A.; Berteau, O.; douki, T. et al. J. Biol. Chem. 2008, 283, 36361.
■ Spore Photoproduct Lyase, recycling or irreversible? Recent experiments suggest No.
HN
N N
NH
O OMe
O O
56
spore photoproduct
HN
N N
NH
O OMe
O O
5
HN
N N
NH
O OMe
O O
5HN
N
Me
N
NH
O OMe
O O
5
Ado-CH2• Ado-CH3
two adjacent thymidines
+ e− + H+
UV-B
Spore photoproduct lyase has to repair DNA lesions that occasionally occur in bacteria germinatedfrom spores.
SAM radical Enzymes
■ 7 Known Irreversible SAM-Glycine-Cysteine Radical Enzymes
Buckel, W.; Golding B. T. Radical Enzymes in Encyclopedia of Radicals in Chemistry, Biology and Materials, 2012, John Wiley & Sons.
■ RNR III (refer to the RNR part)
■ Pyruvate formate lyase
MeHO
O
O
cys-S• MeHOO
OS-cys
HO
O Me
O
cys-S+
cys-SH HO
O Me
O
CoA-S+
H
CoASH
pyruvate formate
■ 2-Oxobutyrate formate lyase (similar to the above)
■ Benzylsuccinate synthase (similar to the above)
■ 2-(1-methylpentyl)succinate synthase
Me
cys-S•
Me
CO2
CO2CO2O2C cys-SH
MeMe
CO2
CO2
fumarateMe
MeMe
MeC-2: Δ BDE ~ 9.6 Kcal/molC-1: Δ BDE ~ 12 Kcal/mol
SAM radical Enzymes
■ 7 Known Irreversible SAM-Glycine-Cysteine Radical Enzymes
Buckel, W.; Golding B. T. Radical Enzymes in Encyclopedia of Radicals in Chemistry, Biology and Materials, 2012, John Wiley & Sons.
■ Glycerol dehydratase (similar to the version of coenzyme B12-dependent enzyme)
■ p-Hydroxyphenylacetate decarboxylase
HOO
O
cys-S• cys-S−
HOO
O
OHO
CH3
CO2 + H+
2 H+
Kolbe-typep-hydroxyphenylacetate
p-cresol p-cresol radical anion
SAM radical Enzymes
■ Irreversible SAM Radical Enzymes: PylB
Gaston, M. A.; Zhang, L.; Green-Church, K. B.; Krzycki, J. A. Nature, 2011, 471, 647.
H2N CO2
NH3
(S)-α-lysine
4 H2N CO2
NH3
4 H2N CO2
NH3H2N
CO2
NH3
Me
(2R, 3R)-3-methylornithine
■ Irreversible SAM Radical Enzymes: HydG
HO
O
ONH3
(S)-tyrosine
Ado-CH2•
O
O
ONH3
- H+
O
O
ONH2
+
dehydroglycine
SAM radical Enzymes
■ Irreversible SAM Radical Enzymes: Sulfur insertion
Atta, M.; Mulliez, E.; Arragain, S. et al. Curr. Opin. Struct. Biol. 2010, 20, 684.
■ Examples of sulfur insertion by SAM radical enzymes
The abstraction of a hydrogen atom bound to carbon is followed by insertion of sulfur derived from a second iron-sulfurcluster of SAM radical enzyme.
S
NHHNH H
O
CO2H
D-biotin
S SProtein
O
protein-bound lipoic acid
HOProtein
O
SMe
HNProtein
O
thiomethylated aspartatein the ribosomal protein S12
SubHAdo-CH2•
SubFe2S2 cluster
Sub−S Pdt
Reactions Involving Ketyl Radicals
■ Backgroup of CoASH and ATP
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.
■ 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.
N
NN
N
NH2
O
OHO
OONH
NH
HSMeMe
OH
OO
coenzyme A (CoASH)
N
NN
N
NH2
O
OHOH
O
PO
O O
adenosine triphosphate (ATP)
PO
PO
O
O
O
PO
PO
PO
O
O
O
O
O
O
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.
Smith, D. M.; Buckel, W.; Zipse, H. Angew. Chem. Int. Ed. 2003, 42, 1867.
CoAS R
O
OH
H
(R)-2-hydroxyacyl-CoA
CoAS R
OpKa ~ 40
CoAS R
O
OH
H
CoAS R
O H
ketyl radical
H+ HOH H+
CoAS R
O
allylic ketyl radicalenoxy radical
pKa ~ 14
e−
2 ADP + 2 Pi
2 ATP + 2 H2Oferredoxin−
(E)-2-enoyl-CoA
Ered ~ −0.9 V
− H2O
Reactions Involving Ketyl Radicals
■ 4-Hydroxybutyryl-CoA dehydratase
Buckel, W.; Golding, B. T. Annu. Rev. Microbiol. 2006, 60, 27.
CoAS
O H
enolateCoAS
O
CoAS
O H
CoAS
O
enoxy radical
H+ HOH
H+
CoAS
O
dienoxy radicalallylic ketyl radical
e−
dienolate
CoAS
O
H
H pKa ~ 40OH
4-hydroxybutyryl-CoA
CoAS Me
O
crotonyl-CoA
H+ H+
OH
OH OH
pKa ~ 14
flavodoxin+ ferredoxin−
pKa ~ 8
N
N
HN
NO
OMe
Me
R
HO
flavodoxin+: FAD
− H2O
Reactions Involving Ketyl Radicals
■ Acyl-CoA Dehydrogenase
Ghisla, S.; Thorp, C. Eur. J. Biochem. 2004, 271, 494.Kitzing, K.; Auweter, S.; Amrhein, N.; Macheroux, P. J. Biol. Chem. 2004, 279, 9451.
CoAS Me
O H
enolate
CoAS Me
O
H
H
butyryl-CoA
CoAS Me
O
CoAS Me
O
H+ e− + H+ e−ketyl radical crotonyl-CoA
FADH•FAD
■ Chorismate Synthase
pKa ~ 8
FADH2
H+
OP
O
OO
H
O CO2
OHO CO2
OH
− HPO42−
FMNH-
O O O O
OP
O
OO
H
O CO2
OH
O O
H
O CO2
OH
O O
+ e-
− PO43− − H+pKa ~ 14
O CO2
OH
O O
− e-
Reactions Involving Ketyl Radicals
■ DNA photolyase
Mees, A.; Klar, T.; Gnau, P. et al. Science, 2004, 306, 1789.
HN
N N
NH
O OMe
O O
56
thymidine dimer
HN
N
Me
N
NH
O OMe
O O
HN
N
Me
N
NH
O OMe
O O
FADH•
FADH−*
290-320 nm UV-B
Me HN
N N
NH
O OMe
O O
Me
+ e−
FADH−
+ e−
two adjacent thymidines
350-450 nm UV-Amethylenetetrahydrogolate
λmax = 380 nm
FADH2-dependent Hydratase
■ 2-Haloacrylate hydratase
Mowafy, A. M.; Kurihara, T.; Kurata, A. et al. Appl. Environ. Microbiol. 2010, 76, 6032.
The hydratase catalyzes the hydration of 2-chlroacrylate to pyruvate and chloride, while a simple hydrationof chloroacrylate would lead to 2-chloro-3-hydroxypropionate.
FADH•
+ e−
FADH−
+ e−
CO2
Cl
H2C CO2
Cl
H3C CO2
Cl
H3C CO2
Cl
+ OH−
H3C CO2
Cl OH
H3C CO2
O + H+ + Cl−
+ H+
CO2
ClHO
2-chlroacrylate
pyruvate2-chloro-3-hydroxypropionate
Reaction of Aromatic Compounds
■ Class I BCR
Boll, M. BioChim. Biophys. Acta. 2005, 1707 , 34.Rees, D. C.; Howard, J. B. Curr. Opin. Chem. Biol. 2000, 4, 559.
2Fdred
SCoA
O
2 ADP + 2 Pi2 ATP + 2 H2O2Fdox
2 H+
SCoA
O
HH
SCoA
OO
SCoA
H
H
HH
SCoA
O
HH −
+ e−
+ H+ + e−
+ H+
■ Protochlorophllide reductase
N N
NN
MeMe
Me Me
HO2C
MgII
OMeO2C
N N
NN
MeMe
Me Me
HO2C
MgII
OMeO2C
NADPH + H+ + hυ
2 Fdred + 2 H+ + 2 ATP
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 givea β-hydroxyalkyl radical that eliminates the C-3 hydroxyl group in the manner of RNR or diol dehydratase.
Tritsch, D.; Hemmerlin, A.; Bach, T. J.; Rohmer, M. FEBS Lett. 2010, 584, 129.
OOH
Me O
OH
P OP
O OO
O2 3 OPP
OH
Me
OH
3 OPPOH
Me
OH
3
''E''+
OPPOH
Me
GcpE GcpE
GcpE − "E"−OH
GcpEOPP
OH
Me
OPP
Me
''E''+
LytB
− "E"−OH+ 2 e−
[Fe4S4]+ [Fe4S4]2+
[Fe4S4]2+ [Fe4S4]+
LytB H+
OPP
Me
Me OPP
MeIPP + DMAPP ''E''+: electrophile
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".
Toraya, T. Cell. Mol. Life Sci. 2000, 57, 106.
HO OHOH
HO O HO OH
glycerol 3-hydroxypropanal 1,3-propanediolH
glycerol dehydratase reductase