Radical Enzymes
PPO N PPO O N O
HO OH HO
Jian Jin MacMillan Group Meeting November 23, 2015 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 Fe Me
cys-S Me Fe S N N cys-S Fe S Fe4S4 cluster FeII S Fe N N S-cys O Me Me N Me HN heme B O N N Me HO2C CO2H NH2 OH N N OH NH HO N 2 O N O O N N O O P O P NH OH 2 O N HO O N OH OH N O O O P O P O O OH flavin adenine dinucleotide (FAD) HO OH OH left half of FAD OH OH flavin mononucleotide (FMN) 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 O O
S OH OH O OH H N NH2 NH2 2 O OH HN NH2 cysteine thiyl radical tyrosine oxy radical glycine carbon radical tryptophan cation radical (cysteinyl) (tyrosyl) (glycyl)
coenzyme B12 / SAM Me Me
Me Me NH2 N N N N N O O N FeIV FeIV N N N S N N S N Me Me Me Me O
HO2C CO2H HO2C CO2H OH OH 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 + H2O + NADP .
■ Oxygen Rebound Mechanism in P450-catalyzed C−H Hydroxylation
O , 2 H+, 2 e− H O Me 2 2 Me
Me Me
N N N O N FeIII FeIV N S N N S N Me Me Me Me
HO2C CO2H HO2C CO2H resting ferric enzyme compound I R−H ROH Me
R Me R
N OH N FeIV N S N Me Me
HO2C 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)
NH2 NH2 PPO OH OH N O O residue residue residue ribonucleotide H H O O HS OH HO OH NH2
OH OH
Tyr-OH Tyr-OH Tyr-OH Tyr-O•
H2O H2O H2O O O O O O MnII MnII MnIII MnIII MnIII MnIV MnIII MnIII O O O O Nrdl O O Nrdl? O O O O
− − − II HO2 HO2 , e (FMNH2) III Mn 2-NrdF Mn 2/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 PPO PPO N N N O O O E441 H H H H O O O HO OH E441 HO OH E441 O O glumate O OH H O NH2 O NH2 O NH2 SH SH SH S pKa ~ 8 SH S
N437 C462 C225 N437 C462 C225 N437 C462 C225
C439 C439 C439
S SH SH
PPO PPO PPO N N N O O O E441 H H H H O O O HO H E441 HO H E441 O H O O OH
O NH2 O NH2 O NH2 S S S S S S
N437 C462 C225 N437 C462 C225 N437 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 AdoCH -Cbl CONH2 2 H2NOC N N Me R Co SH PH H N N Me AdoCH2• + •Cbl Me
H2NOC CONH2 Me Me N Me O NH N Me AdoCH3 + •Cbl HO O S• P• Me P O O Minimal mechanism for coenzyme B -dependent enzymatic reactions O 12
coenzyme B12 (R = 5'-deoxyadenosyl)
Brown, K. L. Chem. Rev. 2005, 105, 2075. Coenzyme B12-Dependent Enzymes
■ Eliminases
H OH OH O R2 2 1 R H O/NH R R1 R2 + 2 3 R1 OH/NH 2 OH/NH2
OH ■ Glycerol dehydratase HO OH HO O
H
OH ■ Propane-1,2-diol dehydratase OH O Me Me H
PPO PPO ■ Ribonucleotide reductases N N O NADPH + H+ O H
HO OH HO
OH ■ Ethanolamine ammonia lyase H2N 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 C R1 R1 H C R1 R1 C
■ Glutamate mutase O2C H O2C H O2C CO2 O2C O2C NH3 NH3 H H H H C NH H H 3 3
■ 2-Methylene-glutarate mutase O C O2C 2 O2C H H H H CH3 O2C
O2C O2C
■ Methylmalonyl-CoA mutase O O2C O O2C O O C 2 SCoA H H H SCoA H C SCoA H 3
■ Isobutyryl-CoA mutase O Me O Me O Me SCoA H H H SCoA H3C SCoA 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
■ Amino Mutases
H N N R1 R1 H N R1 R1 N
■ β-Lysine-5,6-aminomutase NH NH NH3 2 3 CO H2N CO2 2 5 H3C 5 6 6
■ Ornithine-4,5-aminomutase 4 4 CO2 H3C CO2 H2N 5 5 NH3 NH2 NH3 O HO OH HO P O O condensation hydrolyzation HAT Me N HAT coenzyme pyridoxal phosphate
4 CO 4 5 2 CO2 CO2 5 N 5 N 4 N NH3 NH3 NH3 Ar Ar Ar azacyclopropylcarbinyl radical
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.
NH2 N N
N N Me O 5'-deoxyadenosine S OH OH H N S Fe methionine cys-S O ferredoxin or flavodoxin Fe S e O ( E' = −0.5 V) cys-S Fe S
HS Fe Fe4S4 cluster S-cys
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 β- lysine, which means 5-deoxyadenosyl radical is involved and could be regenerated.
NH3 lysine-2,3-aminomutase H2N CO2 H2N CO2 5 5 6 SAM 6 NH3 (S)-α-lysine (S)-β-lysine
O HO OH β-lysine-5,6-aminomutase coenzyme B12 HO P O O
Me N NH2 NH3
CO2 coenzyme pyridoxal phosphate H3C 5 6 ■ DesII
H H Me Me H O H O DesII H H2N HO H H H H OH SAM OH H OTDP O 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 O O O Me Me
HN 5 NH HN 5 NH 6 O N N O O N N O
spore photoproduct Ado-CH2• Ado-CH3
UV-B O O O O Me Me Me + e− + H+ HN NH HN 5 NH 5
O N N O O 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 O cys-S• HO O cys-SH HO H O HO HO Me Me + + S-cys O cys-S Me CoASH O CoA-S Me O O formate pyruvate
■ 2-Oxobutyrate formate lyase (similar to the above)
■ 2-(1-methylpentyl)succinate synthase
CO2 CO2 CO2 cys-S O C Me • Me 2 CO2 cys-SH CO2 Me Me fumarate Me Me 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 O HO HO p-hydroxyphenylacetate Kolbe-type + cys-S• cys-S− CO2 + H
CH3
HO O 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
Me H N CO H2N 4 CO2 H2N 4 CO2 2 2 CO2 H2N NH NH3 NH3 3 NH3 (S)-α-lysine (2R, 3R)-3-methylornithine
■ Irreversible SAM Radical Enzymes: HydG
O O O + Ado-CH2• - H O O + O
NH3 NH3 O NH HO O 2 (S)-tyrosine 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.
Ado-CH2• Fe2S2 cluster SubH Sub Sub−S Pdt
■ Examples of sulfur insertion by SAM radical enzymes
O Me O S O HN NH HO H H Protein Protein S S O HN Protein S CO2H thiomethylated aspartate D-biotin protein-bound lipoic acid 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.
NH2 N N
O O Me Me O O N N coenzyme A (CoASH) HS O O O N N P P H H O OH O O O OH O P O O
NH2 N N
O O O N N O O O O P P P adenosine triphosphate (ATP) O O 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.
O H pKa ~ 40 O − H2O CoAS R CoAS R OH (R)-2-hydroxyacyl-CoA (E)-2-enoyl-CoA
e− 2 ADP + 2 P Ered ~ −0.9 V i
2 ATP + 2 H2O ferredoxin− + H+ HOH H O H O H pKa ~ 14 O
CoAS R CoAS 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 ~ 40 O a − H2O OH CoAS CoAS Me
H pKa ~ 8 4-hydroxybutyryl-CoA O crotonyl-CoA N Me HN H+ H+ O N N Me HO O H O OH R CoAS CoAS flavodoxin+: FAD enolate dienolate
flavodoxin+ ferredoxin− e−
H+ HOH O H pKa ~ 14 O O OH OH CoAS CoAS CoAS
H+ enoxy radical allylic ketyl radical dienoxy radical
Buckel, W.; Golding, B. T. Annu. Rev. Microbiol. 2006, 60, 27. Reactions Involving Ketyl Radicals
■ Acyl-CoA Dehydrogenase
O H O H O O CoAS Me CoAS Me CoAS Me CoAS Me H pKa ~ 8 H+ e− + H+ e− butyryl-CoA enolate ketyl radical crotonyl-CoA H+
FAD FADH• FADH2 ■ Chorismate Synthase
O O O O
H 2− − HPO4
O FMNH- O O CO2 O CO P 2 O O OH OH
+ e- − e-
O O O O O O pKa ~ 14 3− + H − PO4 H − H
O O O CO2 O CO O CO P 2 2 O O OH OH 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 O O Me Me Me Me HN NH − HN 5 NH + e 6 O N N O O N N O
thymidine dimer FADH−* 350-450 nm UV-A 290-320 nm UV-B FADH• methylenetetrahydrogolate λmax = 380 nm FADH− O O O O Me Me Me Me HN NH HN NH
O N N O + e− O N N O
two adjacent thymidines
Mees, A.; Klar, T.; Gnau, P. et al. Science, 2004, 306, 1789. FADH2-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.
Cl + e− Cl
CO2 H2C CO2
2-chlroacrylate + H+ FADH− FADH•
Cl Cl
H3C CO2 + e− H3C CO2 + OH−
Cl Cl OH O + H+ + Cl− HO CO2 H3C CO2 H3C CO2 2-chloro-3-hydroxypropionate pyruvate
Mowafy, A. M.; Kurihara, T.; Kurata, A. et al. Appl. Environ. Microbiol. 2010, 76, 6032. Reaction of Aromatic Compounds
■ Class I BCR
2 ATP + 2 H2O 2 ADP + 2 Pi 2Fd 2Fd red ox O O SCoA SCoA H H 2 H+ H + e− H + H+ O O O + H+ + e− SCoA SCoA SCoA H H − H H
■ Protochlorophllide reductase
Me Me Me Me
N N N N NADPH + H+ + h MgII υ MgII + N N 2 Fdred + 2 H + 2 ATP N N Me Me Me Me
HO2C HO2C
MeO2C O MeO2C 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 O Me Me P O O GcpE GcpE Me O 3 3 P O OPP 3 OPP 2 O OH OH OH OH OH OH + 2+ [Fe4S4] [Fe4S4] ''E''+
GcpE − "E"−OH
Me Me Me LytB GcpE OPP OPP OPP + 2 e− OH OH − "E"−OH 2+ [Fe S ]+ ''E''+ [Fe4S4] 4 4
LytB H+
Me Me + DMAPP IPP ''E''+: electrophile OPP Me OPP
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 glycerol dehydratase reductase HO OH HO O HO OH
H glycerol 3-hydroxypropanal 1,3-propanediol
Toraya, T. Cell. Mol. Life Sci. 2000, 57, 106.