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Radical Enzymes Jian Jin MacMillan Group Meeting November 23, 2015 N O OH HO PPO N O HO PPO

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Page 1: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

Radical Enzymes

Jian JinMacMillan Group Meeting

November 23, 2015

NO

OHHO

PPON

O

HO

PPO

Page 2: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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.

Page 3: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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

Page 4: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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

Page 5: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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

Page 6: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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

Page 7: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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

Page 8: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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

Page 9: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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

Page 10: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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

Page 11: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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

Page 12: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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

Page 13: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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

Page 14: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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.

Page 15: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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.

Page 16: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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

Page 17: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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

Page 18: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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

Page 19: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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

Page 20: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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

Page 21: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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

Page 22: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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

Page 23: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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-

Page 24: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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

Page 25: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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

Page 26: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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

Page 27: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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

Page 28: Radical Enzymes - Princeton University …chemlabs.princeton.edu/.../sites/6/Jian_radical_enzymes.pdfRadical Enzymes Definition The term radical enzyme describes those enzymes that

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