Benzoylformate decarboxylase

 

Benzoylformate decarboxylase is a thiamine diphosphate dependent enzyme. It catalyses the formation of carbon dioxide from bezoylformate and is part of the pathway that synthesises benzoate from (R)-mandelate.

 

Reference Protein and Structure

Sequence
P20906 UniProt (4.1.1.7) IPR029061 (Sequence Homologues) (PDB Homologues)
Biological species
Pseudomonas putida (Bacteria) Uniprot
PDB
1mcz - BENZOYLFORMATE DECARBOXYLASE FROM PSEUDOMONAS PUTIDA COMPLEXED WITH AN INHIBITOR, R-MANDELATE (2.8 Å) PDBe PDBsum 1mcz
Catalytic CATH Domains
3.40.50.1220 CATHdb 3.40.50.970 CATHdb (see all for 1mcz)
Cofactors
Thiamine(1+) diphosphate(3-) (1), Magnesium(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:4.1.1.7)

phenylglyoxylate
CHEBI:36656ChEBI
+
hydron
CHEBI:15378ChEBI
→
carbon dioxide
CHEBI:16526ChEBI
+
benzaldehyde
CHEBI:17169ChEBI
Alternative enzyme names: Phenylglyoxylate decarboxylase, Benzoylformate carboxy-lyase,

Enzyme Mechanism

Introduction

This alternative mechanism is based off more up to date computational experiments (26/06/2018), which used density functional theory calculations. The mechanism begins with intramolecular proton transfer within the cofactor which activates it for nucleophilic attack on phenylglyoxylate. This results in an oxyanion being formed which can accept a proton from the His70/Glu28 proton relay system. Carbon dioxide is then eliminated with TDP acting as an electron sink. TDP then tautomerizes, this is facilitated by another intramolecular proton transfer. The oxyanion is regenerated by the proton relay system, and its collapse leads to TDP being cleaved and the product being formed. TDP then undergoes a final intramolecular proton transfer which results in the cofactor being regenerated in its native state. See other mechanism for further references.

Catalytic Residues Roles

UniProt PDB* (1mcz)
His70, Glu28 His70E, Glu28E Part of a proton relay system throughout the mechanism. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, proton relay, electrostatic stabiliser
Gly25 (main-N), Ser26 Gly25E (main-N), Ser26E Stabilizes the reactant via hydrogen bonding. hydrogen bond donor, electrostatic stabiliser
*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, bimolecular nucleophilic addition, intermediate formation, proton relay, decarboxylation, intramolecular elimination, tautomerisation (not keto-enol), overall product formed, intermediate terminated, native state of cofactor regenerated

References

  1. Planas F et al. (2018), Front Chem, 6, 205-. A Theoretical Study of the Benzoylformate Decarboxylase Reaction Mechanism. DOI:10.3389/fchem.2018.00205. PMID:29998094.

Step 1. There is intramolecular proton transfer within the cofactor, resulting in an ylide being formed.

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Catalytic Residues Roles

Residue Roles
Gly25E (main-N) hydrogen bond donor
Ser26E hydrogen bond donor
Glu28E hydrogen bond donor
His70E hydrogen bond donor, hydrogen bond acceptor
Gly25E (main-N) electrostatic stabiliser
Ser26E electrostatic stabiliser
Glu28E electrostatic stabiliser
His70E electrostatic stabiliser

Chemical Components

proton transfer, cofactor used

Step 2. The carboanion of the ylide performs a nucleophilic attack on the carbonyl carbon resulting in an oxyanion intermediate being formed.

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Catalytic Residues Roles

Residue Roles
Gly25E (main-N) hydrogen bond donor
Ser26E hydrogen bond donor
Glu28E hydrogen bond donor
His70E hydrogen bond donor, hydrogen bond acceptor
Gly25E (main-N) electrostatic stabiliser
Ser26E electrostatic stabiliser
Glu28E electrostatic stabiliser
His70E electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic addition, intermediate formation

Step 3. The oxyanion accepts a proton from His70, which accepts a proton from Glu28 in a proton relay.

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Catalytic Residues Roles

Residue Roles
Gly25E (main-N) hydrogen bond donor
Ser26E hydrogen bond donor
Glu28E hydrogen bond donor
His70E hydrogen bond donor
Gly25E (main-N) electrostatic stabiliser
Ser26E electrostatic stabiliser
Glu28E electrostatic stabiliser
His70E electrostatic stabiliser
His70E proton relay
His70E proton acceptor
Glu28E proton donor
His70E proton donor

Chemical Components

proton transfer, proton relay

Step 4. Carbon dioxide is eliminated from the intermediate, with TDP acting as an electron sink.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly25E (main-N) hydrogen bond donor
Ser26E hydrogen bond donor
Glu28E hydrogen bond donor
His70E hydrogen bond donor
Gly25E (main-N) electrostatic stabiliser
Ser26E electrostatic stabiliser
Glu28E electrostatic stabiliser
His70E electrostatic stabiliser, hydrogen bond acceptor

Chemical Components

decarboxylation, ingold: intramolecular elimination

Step 5. TDP initiates a double bond rearrangement which results in an intramolecular proton transfer within the intermediate.

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Catalytic Residues Roles

Residue Roles
Gly25E (main-N) hydrogen bond donor
Ser26E hydrogen bond donor
Glu28E hydrogen bond donor
His70E hydrogen bond donor
Gly25E (main-N) electrostatic stabiliser
Ser26E electrostatic stabiliser
Glu28E electrostatic stabiliser
His70E electrostatic stabiliser, hydrogen bond acceptor

Chemical Components

tautomerisation (not keto-enol), proton relay

Step 6. The hydroxyl oxygen is deprotonated by the Glu28/His70 proton relay system, resulting in the formation of another oxyanion.

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Catalytic Residues Roles

Residue Roles
Gly25E (main-N) hydrogen bond donor
Ser26E hydrogen bond donor
Glu28E hydrogen bond donor
His70E hydrogen bond donor
Gly25E (main-N) electrostatic stabiliser
Ser26E electrostatic stabiliser
Glu28E electrostatic stabiliser
His70E electrostatic stabiliser, hydrogen bond acceptor
His70E proton relay
Glu28E proton acceptor
His70E proton donor, proton acceptor

Chemical Components

proton transfer, proton relay

Step 7. The imine nitrogen of the cofactor then accepts a proton from the relay system.

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Catalytic Residues Roles

Residue Roles
Gly25E (main-N) hydrogen bond donor
Ser26E hydrogen bond donor
Glu28E hydrogen bond donor
His70E hydrogen bond donor
Gly25E (main-N) electrostatic stabiliser
Ser26E electrostatic stabiliser
Glu28E electrostatic stabiliser
His70E electrostatic stabiliser, hydrogen bond acceptor
His70E proton relay
Glu28E proton donor
His70E proton acceptor, proton donor

Chemical Components

proton relay, proton transfer

Step 8. The collapse of the oxyanion leads to the elimination of TDP and the formation of the product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly25E (main-N) hydrogen bond donor
Ser26E hydrogen bond donor
Glu28E hydrogen bond donor
His70E hydrogen bond donor
Gly25E (main-N) electrostatic stabiliser
Ser26E electrostatic stabiliser
Glu28E electrostatic stabiliser
His70E electrostatic stabiliser, hydrogen bond acceptor

Chemical Components

overall product formed, ingold: intramolecular elimination, intermediate terminated

Step 9. The cofactor is regenerated in its native state via an intramolecular proton transfer.

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Catalytic Residues Roles

Residue Roles

Chemical Components

proton relay, native state of cofactor regenerated
Download: Image, Marvin File

Step 1. There is intramolecular proton transfer within the cofactor, resulting in an ylide being formed.

Step 2. The carboanion of the ylide performs a nucleophilic attack on the carbonyl carbon resulting in an oxyanion intermediate being formed.

Step 3. The oxyanion accepts a proton from His70, which accepts a proton from Glu28 in a proton relay.

Step 4. Carbon dioxide is eliminated from the intermediate, with TDP acting as an electron sink.

Step 5. TDP initiates a double bond rearrangement which results in an intramolecular proton transfer within the intermediate.

Step 6. The hydroxyl oxygen is deprotonated by the Glu28/His70 proton relay system, resulting in the formation of another oxyanion.

Step 7. The imine nitrogen of the cofactor then accepts a proton from the relay system.

Step 8. The collapse of the oxyanion leads to the elimination of TDP and the formation of the product.

Step 9. The cofactor is regenerated in its native state via an intramolecular proton transfer.

Products.

Introduction

In this 3rd proposed mechanism, based off the same 2018 computational study, decarboxylation occurs immediately after the formation of the substrate-cofactor intermediate. The difference barrier energies between these two mechanisms is not sufficiently high to conclude which is the more accurate. See the other mechanisms for the references.

Catalytic Residues Roles

UniProt PDB* (1mcz)
*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

cofactor used, proton transfer, intermediate formation, bimolecular nucleophilic addition, decarboxylation, intramolecular elimination, proton relay, tautomerisation (not keto-enol), intermediate terminated, overall product formed, native state of cofactor regenerated

References

Step 1. There is intramolecular proton transfer within the cofactor, resulting in an ylide being formed.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His70E electrostatic stabiliser
Glu28E electrostatic stabiliser
Ser26E electrostatic stabiliser
Gly25E (main-N) electrostatic stabiliser
His70E hydrogen bond acceptor, hydrogen bond donor
Glu28E hydrogen bond donor
Ser26E hydrogen bond donor
Gly25E (main-N) hydrogen bond donor

Chemical Components

cofactor used, proton transfer

Step 2. The carboanion of the ylide performs a nucleophilic attack on the carbonyl carbon resulting in an oxyanion intermediate being formed.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His70E electrostatic stabiliser
Glu28E electrostatic stabiliser
Ser26E electrostatic stabiliser
Gly25E (main-N) electrostatic stabiliser
His70E hydrogen bond acceptor, hydrogen bond donor
Glu28E hydrogen bond donor
Ser26E hydrogen bond donor
Gly25E (main-N) hydrogen bond donor

Chemical Components

intermediate formation, ingold: bimolecular nucleophilic addition

Step 3. Carbon dioxide is eliminated from the intermediate, with TDP acting as an electron sink.

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Catalytic Residues Roles

Residue Roles
Gly25E (main-N) electrostatic stabiliser
Ser26E electrostatic stabiliser
Glu28E electrostatic stabiliser
His70E electrostatic stabiliser
Gly25E (main-N) hydrogen bond donor
Ser26E hydrogen bond donor
Glu28E hydrogen bond donor
His70E hydrogen bond donor
His70E hydrogen bond acceptor

Chemical Components

decarboxylation, ingold: intramolecular elimination

Step 4. The enolate oxygen accepts a proton from the His70/Glu28 proton relay system.

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Catalytic Residues Roles

Residue Roles
Gly25E (main-N) hydrogen bond donor
Ser26E hydrogen bond donor
Glu28E hydrogen bond donor
His70E hydrogen bond donor
Gly25E (main-N) electrostatic stabiliser
Ser26E electrostatic stabiliser
Glu28E electrostatic stabiliser
His70E electrostatic stabiliser, hydrogen bond acceptor
His70E proton relay
Glu28E proton donor
His70E proton acceptor, proton donor

Chemical Components

proton relay, proton transfer

Step 5. TDP initiates a double bond rearrangement which results in an intramolecular proton transfer within the intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His70E hydrogen bond acceptor, electrostatic stabiliser
Glu28E electrostatic stabiliser
Ser26E electrostatic stabiliser
Gly25E (main-N) electrostatic stabiliser
His70E hydrogen bond donor
Glu28E hydrogen bond donor
Ser26E hydrogen bond donor
Gly25E (main-N) hydrogen bond donor

Chemical Components

proton relay, tautomerisation (not keto-enol)

Step 6. The hydroxyl oxygen is deprotonated by the Glu28/His70 proton relay system, resulting in the formation of another oxyanion.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His70E proton relay
His70E hydrogen bond acceptor, electrostatic stabiliser
Glu28E electrostatic stabiliser
Ser26E electrostatic stabiliser
Gly25E (main-N) electrostatic stabiliser
His70E hydrogen bond donor
Glu28E hydrogen bond donor
Ser26E hydrogen bond donor
Gly25E (main-N) hydrogen bond donor
His70E proton donor
Glu28E proton acceptor
His70E proton acceptor

Chemical Components

proton relay, proton transfer

Step 7. The imine nitrogen of the cofactor then accepts a proton from the relay system.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His70E proton relay
His70E hydrogen bond acceptor, electrostatic stabiliser
Glu28E electrostatic stabiliser
Ser26E electrostatic stabiliser
Gly25E (main-N) electrostatic stabiliser
His70E hydrogen bond donor
Glu28E hydrogen bond donor
Ser26E hydrogen bond donor
Gly25E (main-N) hydrogen bond donor
His70E proton acceptor
Glu28E proton donor
His70E proton donor

Chemical Components

proton transfer, proton relay

Step 8. The collapse of the oxyanion leads to the elimination of TDP and the formation of the product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His70E hydrogen bond acceptor, electrostatic stabiliser
Glu28E electrostatic stabiliser
Ser26E electrostatic stabiliser
Gly25E (main-N) electrostatic stabiliser
His70E hydrogen bond donor
Glu28E hydrogen bond donor
Ser26E hydrogen bond donor
Gly25E (main-N) hydrogen bond donor

Chemical Components

intermediate terminated, ingold: intramolecular elimination, overall product formed

Step 9. The cofactor is regenerated in its native state via an intramolecular proton transfer.

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Catalytic Residues Roles

Residue Roles

Chemical Components

native state of cofactor regenerated, proton relay
Download: Image, Marvin File

Step 1. There is intramolecular proton transfer within the cofactor, resulting in an ylide being formed.

Step 2. The carboanion of the ylide performs a nucleophilic attack on the carbonyl carbon resulting in an oxyanion intermediate being formed.

Step 3. Carbon dioxide is eliminated from the intermediate, with TDP acting as an electron sink.

Step 4. The enolate oxygen accepts a proton from the His70/Glu28 proton relay system.

Step 5. TDP initiates a double bond rearrangement which results in an intramolecular proton transfer within the intermediate.

Step 6. The hydroxyl oxygen is deprotonated by the Glu28/His70 proton relay system, resulting in the formation of another oxyanion.

Step 7. The imine nitrogen of the cofactor then accepts a proton from the relay system.

Step 8. The collapse of the oxyanion leads to the elimination of TDP and the formation of the product.

Step 9. The cofactor is regenerated in its native state via an intramolecular proton transfer.

Products.

Introduction

Glu47E deprotonates the thiamine diphosphate cofactor, which initiates double bond rearrangement that results in the deprotonation of the N=CH-S group, activating the thiamine diphosphate cofactor. The carbanion of thiamine diphosphate attacks the carbonyl carbon of alpha-oxo-benzeneacetic acid in a nucleophilic addition. The formed oxyanion deprotonates His70E. Carbon dioxide eliminates from the covalently attached aromatic intermediate. Thiamine diphosphate acts as an electron sink. Thiamine diphosphate initiates a double bond rearrangement, which results in the intermediate being protonated by His281A. His70E deprotonates the hydroxide of the intermediate, which initiates an elimination which results in a reformation of the carbanionic activated cofactor and the benzaldehyde product. The carbanion of the thiamine diphosphate cofactor deprotonates the adjacent amine, which initiates double bond rearrangement that results in the deprotonation of Glu47E. His281A deprotonates water.

Catalytic Residues Roles

UniProt PDB* (1mcz)
Glu47 Glu47E Acts as a general acid/base, important for activating the thiamine diphosphate cofactor. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Glu28 Glu28E Activates His70B. hydrogen bond acceptor, electrostatic stabiliser
Ser26 Ser26E Activates His281A hydrogen bond acceptor, electrostatic stabiliser
His70, His281 His70E, His281F Acts as a general acid/base. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Gly401 (main-C) Gly401F (main-C) Stabilises the reactive intermediates of the thiamine-diphosphate cofactor. hydrogen bond acceptor, electrostatic stabiliser
*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, assisted tautomerisation (not keto-enol), cofactor used, inferred reaction step, bimolecular nucleophilic addition, aldol addition, overall reactant used, intermediate formation, unimolecular elimination by the conjugate base, overall product formed, intermediate collapse, bimolecular elimination, native state of cofactor regenerated, native state of enzyme regenerated

References

  1. Polovnikova ES et al. (2003), Biochemistry, 42, 1820-1830. Structural and Kinetic Analysis of Catalysis by a Thiamin Diphosphate-Dependent Enzyme, Benzoylformate Decarboxylase†. DOI:10.1021/bi026490k. PMID:12590569.
  2. Hasson MS et al. (1998), Biochemistry, 37, 9918-9930. The Crystal Structure of Benzoylformate Decarboxylase at 1.6 Å Resolution:  Diversity of Catalytic Residues in Thiamin Diphosphate-Dependent Enzymes†,‡. DOI:10.1021/bi973047e. PMID:9665697.

Step 1. Glu47E deprotonates the thiamine diphosphate cofactor, which initiates double bond rearrangement that results in the deprotonation of the N=CH-S group, activating the cofactor.

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Catalytic Residues Roles

Residue Roles
Glu47E hydrogen bond acceptor
Gly401F (main-C) hydrogen bond acceptor, electrostatic stabiliser
Ser26E hydrogen bond acceptor, electrostatic stabiliser
His281F hydrogen bond donor
His70E hydrogen bond donor
Glu28E hydrogen bond acceptor, electrostatic stabiliser
Glu47E proton acceptor

Chemical Components

proton transfer, assisted tautomerisation (not keto-enol), cofactor used, inferred reaction step

Step 2. The carbanion of thiamine diphosphate attacks the carbonyl carbon of alpha-oxo-benzeneacetic acid in a nucleophilic addition. The formed oxyanion deprotonates His70E

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Catalytic Residues Roles

Residue Roles
Glu47E hydrogen bond donor
Gly401F (main-C) hydrogen bond acceptor, electrostatic stabiliser
Ser26E hydrogen bond acceptor, electrostatic stabiliser
His281F hydrogen bond donor
His70E hydrogen bond donor
Glu28E hydrogen bond acceptor, electrostatic stabiliser
His70E proton donor

Chemical Components

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

Step 3. Carbon dioxide eliminates from the covalently attached aromatic intermediate. Thiamine diphosphate acts as an electron sink.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu47E hydrogen bond donor
Gly401F (main-C) hydrogen bond acceptor, electrostatic stabiliser
Ser26E hydrogen bond acceptor, electrostatic stabiliser
His281F hydrogen bond donor
His70E hydrogen bond donor
Glu28E hydrogen bond acceptor, electrostatic stabiliser

Chemical Components

ingold: unimolecular elimination by the conjugate base, assisted tautomerisation (not keto-enol), overall product formed, intermediate formation, intermediate collapse

Step 4. Thiamine diphosphate initiates a double bond rearrangement, which results in the intermediate being protonated by His281A.

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Catalytic Residues Roles

Residue Roles
Glu47E hydrogen bond donor
Gly401F (main-C) hydrogen bond acceptor, electrostatic stabiliser
Ser26E hydrogen bond acceptor, electrostatic stabiliser
His281F hydrogen bond donor
His70E hydrogen bond donor
Glu28E hydrogen bond acceptor, electrostatic stabiliser
His281F proton donor

Chemical Components

proton transfer, assisted tautomerisation (not keto-enol), intermediate formation

Step 5. His70E deprotonates the hydroxide of the intermediate, which initiates an elimination which results in a reformation of the carbanionic activated cofactor and the benzaldehyde product.

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Catalytic Residues Roles

Residue Roles
Glu47E hydrogen bond donor
Gly401F (main-C) hydrogen bond acceptor, electrostatic stabiliser
Ser26E hydrogen bond acceptor, electrostatic stabiliser
His281F hydrogen bond donor
His70E hydrogen bond acceptor, hydrogen bond donor
Glu28E hydrogen bond acceptor, electrostatic stabiliser
His70E proton acceptor

Chemical Components

ingold: bimolecular elimination, intermediate collapse, overall product formed

Step 6. The carbanion of the thiamine diphosphate cofactor deprotonates the adjacent amine, which initiates double bond rearrangement that results in the deprotonation of Glu47E. His281A deprotonates water.

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Catalytic Residues Roles

Residue Roles
Glu47E hydrogen bond donor
Gly401F (main-C) hydrogen bond acceptor, electrostatic stabiliser
Ser26E hydrogen bond acceptor, electrostatic stabiliser
His281F hydrogen bond donor, hydrogen bond acceptor
His70E hydrogen bond donor
Glu28E hydrogen bond acceptor, electrostatic stabiliser
His281F proton acceptor
Glu47E proton donor

Chemical Components

proton transfer, assisted tautomerisation (not keto-enol), native state of cofactor regenerated, native state of enzyme regenerated, inferred reaction step
Download: Image, Marvin File

Step 1. Glu47E deprotonates the thiamine diphosphate cofactor, which initiates double bond rearrangement that results in the deprotonation of the N=CH-S group, activating the cofactor.

Step 2. The carbanion of thiamine diphosphate attacks the carbonyl carbon of alpha-oxo-benzeneacetic acid in a nucleophilic addition. The formed oxyanion deprotonates His70E

Step 3. Carbon dioxide eliminates from the covalently attached aromatic intermediate. Thiamine diphosphate acts as an electron sink.

Step 4. Thiamine diphosphate initiates a double bond rearrangement, which results in the intermediate being protonated by His281A.

Step 5. His70E deprotonates the hydroxide of the intermediate, which initiates an elimination which results in a reformation of the carbanionic activated cofactor and the benzaldehyde product.

Step 6. The carbanion of the thiamine diphosphate cofactor deprotonates the adjacent amine, which initiates double bond rearrangement that results in the deprotonation of Glu47E. His281A deprotonates water.

Products.

Contributors

Gemma L. Holliday, Alex Gutteridge, Craig Porter, James Willey