Pyruvate dehydrogenase (cytochrome)

 

Catalyses the formation of acetate and carbon dioxide from pyruvate with the concomittant reduction of ubiquinone. This protein requires thiamine phosphate and FAD for activity. The bacterial enzyme is located on the inner surface of the cytoplasmic membrane and coupled to the respiratory chain via ubiquinone. It does not accept menaquinone as an acceptor. In Escherichia coli this is the primary complex responsible for aerobic growth, pyruvate oxidase (MACiE:274) acts as a backup system.

 

Reference Protein and Structure

Sequence
P07003 UniProt (1.2.5.1) IPR029061 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
3ey9 - Structural basis for membrane binding and catalytic activation of the peripheral membrane enzyme pyruvate oxidase from Escherichia coli (2.9 Å) PDBe PDBsum 3ey9
Catalytic CATH Domains
3.40.50.970 CATHdb (see all for 3ey9)
Cofactors
Fadh2(2-) (1), Thiamine(1+) diphosphate(3-) (1), Magnesium(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:1.2.5.1)

ubiquinones
CHEBI:16389ChEBI
+
water
CHEBI:15377ChEBI
+
pyruvate
CHEBI:15361ChEBI
→
carbon dioxide
CHEBI:16526ChEBI
+
ubiquinol
CHEBI:17976ChEBI
+
acetate
CHEBI:30089ChEBI
Alternative enzyme names: Pyruvate dehydrogenase, Pyruvic dehydrogenase, Pyruvic (cytochrome b1) dehydrogenase, Pyruvate:ubiquinone-8-oxidoreductase, Pyruvate oxidase, Pyruvate dehydrogenase (cytochrome),

Enzyme Mechanism

Introduction

Glu50' deprotonates the thiamine diphosphate cofactor at the N1 position. This initiates double bond rearrangement which results in the deprotonation of the N=CH-S group. This activates the cofactor towards electrophilic attack. The carbanion of thiamine diphosphate initiates a nucleophilic attack on the carbonyl carbon of pyruvate in an addition reaction. The conjugated double bond system of the cofactor undergoes rearrangement which results in the deprotonation of Glu50'. The covalently bound pyruvate undergoes decarboxylation. A single electron is transferred from the high energy thamine diphosphate enamine intermediate to the FAD cofactor. This results in bond order rearrangement and deprotonation of the alcohol group present on the intermediate. Tautomerisation of the radical intermediate occurs. The thiamine ring nitrogen acts as an electron sink in the formation of a second radical tautomer. Water acts as a nucleophile towards the neutral radical thiamine diphosphate-pyruvate adduct. The hydrolysis product delivers a second reducing equivalent to the FAD cofactor. The tetrahedral intermediate collapses, eliminating acetate and regenerating the carbanionic form of thiamine diphosphate. The thiamine diphosphate cofactor is regenerated on deprotonation of the pyruvate product. Ubiquinone receives one reducing equivalent from the reduced FAD cofactor. The second reducing equivalent is transferred to the bound ubiquinone. This forms the electron transport agent ubiquinol and regenerates the FAD cofactor.

Catalytic Residues Roles

UniProt PDB* (3ey9)
Gln113, Val380 Gln113B, Val380A The steric and electrostatic interactions between the intermediate and residues Val380 and Gln133, respectively holds the TPP cofactor in a high energy conformation which also contributes to enhanced reactivity. promote heterolysis, hydrogen bond acceptor
Phe465 Phe465A Phe465 has been shown by kinetic analysis to be crucial for the efficient transfer of electrons between the thiamine diphosphate and FAD cofactors [PMID:18988747]. polar/non-polar interaction, electrostatic destabiliser, promote heterolysis, radical stabiliser, steric role
Glu50 Glu50B Acts as a general acid/base in the activation of the thiamine diphosphate cofactor. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, activator, increase acidity, promote heterolysis
Phe112 Phe112B Helps stabilise the high energy radical intermediates formed during the course of the reaction. radical stabiliser, promote heterolysis, electrostatic destabiliser, polar/non-polar interaction
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, cofactor used, intermediate formation, bimolecular nucleophilic addition, overall reactant used, intramolecular elimination, decarboxylation, intermediate collapse, overall product formed, electron transfer, redox reaction, rate-determining step, tautomerisation (not keto-enol), inferred reaction step, unimolecular elimination by the conjugate base, native state of cofactor regenerated, intermediate terminated, native state of enzyme regenerated

References

  1. Neumann P et al. (2008), Proc Natl Acad Sci U S A, 105, 17390-17395. Structural basis for membrane binding and catalytic activation of the peripheral membrane enzyme pyruvate oxidase from Escherichia coli. DOI:10.1073/pnas.0805027105. PMID:18988747.
  2. Tittmann K (2009), FEBS J, 276, 2454-2468. Reaction mechanisms of thiamin diphosphate enzymes: redox reactions. DOI:10.1111/j.1742-4658.2009.06966.x. PMID:19476487.
  3. Chang YY et al. (2000), Biochem J, 352, 717-724. Conversion of Escherichia coli pyruvate oxidase to an ‘α-ketobutyrate oxidase’. DOI:10.1042/bj3520717. PMID:11104678.
  4. Tittmann K et al. (1998), J Biol Chem, 273, 12929-12934. Activation of Thiamin Diphosphate and FAD in the Phosphatedependent Pyruvate Oxidase fromLactobacillus plantarum. DOI:10.1074/jbc.273.21.12929. PMID:9582325.

Step 1. Glu50' deprotonates the thiamine diphosphate cofactor at the N1 position. This initiates double bond rearrangement which results in the deprotonation of the N=CH-S group. This activates the cofactor towards electrophilic attack.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gln113B hydrogen bond acceptor
Glu50B activator, hydrogen bond acceptor, increase acidity
Val380A van der waals interaction
Glu50B proton acceptor

Chemical Components

proton transfer, cofactor used, intermediate formation

Step 2. The thiamine diphosphate cofactor maintains the canonical V conformation [PMID:18988747]. The carbanion of thiamine diphosphate initiates a nucleophilic attack on the carbonyl carbon of pyruvate in an addition reaction. The conjugated double bond system of the cofactor undergoes rearrangement which results in the deprotonation of Glu50'.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gln113B hydrogen bond acceptor
Glu50B activator, hydrogen bond donor
Val380A van der waals interaction
Glu50B proton donor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, overall reactant used, intermediate formation

Step 3. The covalently bound pyruvate undergoes decarboxylation. The interactions between the thiamine diphosphate-pyruvate adduct and surrounding residues direct a reactive conformation which lowers the reaction barrier towards decarboxylation [PMID:18988747, PMID:11104678]. This step is not thought to be rate determining [PMID:19476487].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe465A electrostatic destabiliser, promote heterolysis
Phe112B electrostatic destabiliser, promote heterolysis, polar/non-polar interaction
Gln113B hydrogen bond acceptor, promote heterolysis
Glu50B hydrogen bond donor, promote heterolysis
Val380A steric role, electrostatic destabiliser, promote heterolysis

Chemical Components

ingold: intramolecular elimination, decarboxylation, intermediate collapse, intermediate formation, overall product formed

Step 4. A single electron is transferred from the high energy thamine diphosphate enamine intermediate to the FAD cofactor. This results in bond order rearrangement and deprotonation of the alcohol group present on the intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe465A radical stabiliser, polar/non-polar interaction
Phe112B radical stabiliser, polar/non-polar interaction
Gln113B hydrogen bond acceptor
Glu50B hydrogen bond donor
Val380A radical stabiliser

Chemical Components

electron transfer, proton transfer, redox reaction, cofactor used, intermediate formation, rate-determining step

Step 5. Tautomerisation of the radical intermediate occurs. The product of the rearrangement is required in the next reaction step [PMID:19476487].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe465A radical stabiliser, polar/non-polar interaction
Phe112B radical stabiliser, polar/non-polar interaction
Gln113B hydrogen bond acceptor
Glu50B hydrogen bond donor
Val380A radical stabiliser

Chemical Components

tautomerisation (not keto-enol), intermediate formation, inferred reaction step

Step 6. The thiamine ring nitrogen acts as an electron sink in the formation of a second radical tautomer.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe465A radical stabiliser, polar/non-polar interaction
Phe112B radical stabiliser, polar/non-polar interaction
Gln113B hydrogen bond acceptor
Glu50B hydrogen bond donor
Val380A radical stabiliser

Chemical Components

tautomerisation (not keto-enol), intermediate formation

Step 7. Water acts as a nucleophile towards the neutral radical thiamine diphosphate-pyruvate adduct.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe465A radical stabiliser, polar/non-polar interaction
Phe112B radical stabiliser, polar/non-polar interaction
Gln113B hydrogen bond acceptor
Glu50B hydrogen bond donor
Val380A radical stabiliser

Chemical Components

ingold: bimolecular nucleophilic addition, overall reactant used, intermediate formation

Step 8. The hydrolysis product delivers a second reducing equivalent to the FAD cofactor.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe465A radical stabiliser, polar/non-polar interaction
Phe112B radical stabiliser, polar/non-polar interaction
Gln113B hydrogen bond acceptor
Glu50B hydrogen bond donor
Val380A radical stabiliser

Chemical Components

electron transfer, proton transfer, redox reaction, intermediate formation

Step 9. The tetrahedral intermediate collapses, eliminating acetate and regenerating the carbanionic form of thiamine diphosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe465A radical stabiliser, polar/non-polar interaction
Phe112B radical stabiliser, polar/non-polar interaction
Gln113B hydrogen bond acceptor
Glu50B hydrogen bond donor
Val380A radical stabiliser

Chemical Components

ingold: unimolecular elimination by the conjugate base, intermediate collapse, intermediate formation, overall product formed

Step 10. The thiamine diphosphate cofactor is regenerated on deprotonation of the pyruvate product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe465A radical stabiliser, polar/non-polar interaction
Phe112B radical stabiliser, polar/non-polar interaction
Gln113B hydrogen bond acceptor
Glu50B hydrogen bond donor
Val380A radical stabiliser

Chemical Components

proton transfer, native state of cofactor regenerated, overall product formed, intermediate terminated

Step 11. The reduction of FAD initiates a conformational rearrangement of the C terminus. This results in the exposure of a high lipid affinity binding site.
Pyruvate oxidase from E. coli does not use molecular oxygen as a final electron acceptor, unlike its Lactobacillus plantarum analogue [PMID:18988747, PMID:11104678]. Once pyruvate has adhered to a biological membrane, the prior structural rearrangement now allows two electrons to be shuttled from reduced flavin to the membrane bound ubiquinone electron carrier [PMID:18988747]. Ubiquinone receives one reducing equivalent from the reduced FAD cofactor.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe465A steric role, polar/non-polar interaction
Gln113B hydrogen bond acceptor
Glu50B hydrogen bond acceptor

Chemical Components

electron transfer, proton transfer, redox reaction, overall reactant used, intermediate formation

Step 12. The second reducing equivalent is transferred to the bound ubiquinone. This forms the electron transport agent ubiquinol and regenerates the FAD cofactor. Ubiquinol acts as a mobile electron carrier in the electron transport chain [PMID:18988747].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe465A steric role, polar/non-polar interaction
Gln113B hydrogen bond acceptor
Glu50B hydrogen bond acceptor

Chemical Components

electron transfer, proton transfer, redox reaction, native state of cofactor regenerated, overall product formed, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Glu50' deprotonates the thiamine diphosphate cofactor at the N1 position. This initiates double bond rearrangement which results in the deprotonation of the N=CH-S group. This activates the cofactor towards electrophilic attack.

Step 2. The thiamine diphosphate cofactor maintains the canonical V conformation [PMID:18988747]. The carbanion of thiamine diphosphate initiates a nucleophilic attack on the carbonyl carbon of pyruvate in an addition reaction. The conjugated double bond system of the cofactor undergoes rearrangement which results in the deprotonation of Glu50'.

Step 3. The covalently bound pyruvate undergoes decarboxylation. The interactions between the thiamine diphosphate-pyruvate adduct and surrounding residues direct a reactive conformation which lowers the reaction barrier towards decarboxylation [PMID:18988747, PMID:11104678]. This step is not thought to be rate determining [PMID:19476487].

Step 4. A single electron is transferred from the high energy thamine diphosphate enamine intermediate to the FAD cofactor. This results in bond order rearrangement and deprotonation of the alcohol group present on the intermediate.

Step 5. Tautomerisation of the radical intermediate occurs. The product of the rearrangement is required in the next reaction step [PMID:19476487].

Step 6. The thiamine ring nitrogen acts as an electron sink in the formation of a second radical tautomer.

Step 7. Water acts as a nucleophile towards the neutral radical thiamine diphosphate-pyruvate adduct.

Step 8. The hydrolysis product delivers a second reducing equivalent to the FAD cofactor.

Step 9. The tetrahedral intermediate collapses, eliminating acetate and regenerating the carbanionic form of thiamine diphosphate.

Step 10. The thiamine diphosphate cofactor is regenerated on deprotonation of the pyruvate product.

Step 11. The reduction of FAD initiates a conformational rearrangement of the C terminus. This results in the exposure of a high lipid affinity binding site.
Pyruvate oxidase from E. coli does not use molecular oxygen as a final electron acceptor, unlike its Lactobacillus plantarum analogue [PMID:18988747, PMID:11104678]. Once pyruvate has adhered to a biological membrane, the prior structural rearrangement now allows two electrons to be shuttled from reduced flavin to the membrane bound ubiquinone electron carrier [PMID:18988747]. Ubiquinone receives one reducing equivalent from the reduced FAD cofactor.

Step 12. The second reducing equivalent is transferred to the bound ubiquinone. This forms the electron transport agent ubiquinol and regenerates the FAD cofactor. Ubiquinol acts as a mobile electron carrier in the electron transport chain [PMID:18988747].

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday