Benzoin aldolase

 

Thiamine diphosphate dependent benzaldehyde lyase (BAL) from Pseudomonas fluorescens catalyses the cleavage of (R)-benzoin producing two molecules of benzaldehyde. This allows the bacterium to grow on (R)-benzoin as its only carbon and energy source.

The reverse reaction is catalysed by benzoylformate decarboxylase (BFD). The X-ray structure of BAL was compared to that of BFD and pyruvate decarboxylase (PDC) in order to determine which residues are likely to have catalytic roles and should be analysed by point mutations.

BAL looses its catalytic activity upon treatment with EDTA but activity can be restored by addition of 1millimolar MgCl2, MnSO4 or CaSO4.

 

Reference Protein and Structure

Sequence
Q9F4L3 UniProt IPR029061 (Sequence Homologues) (PDB Homologues)
Biological species
Pseudomonas fluorescens (Bacteria) Uniprot
PDB
2ag0 - Crystal structure of Benzaldehyde lyase (BAL)- native (2.58 Å) PDBe PDBsum 2ag0
Catalytic CATH Domains
3.40.50.970 CATHdb (see all for 2ag0)
Cofactors
Thiamine(1+) diphosphate(3-) (1), Magnesium(2+) (1), Water (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:4.1.2.38)

benzoin
CHEBI:17682ChEBI
→
benzaldehyde
CHEBI:17169ChEBI
Alternative enzyme names: Benzaldehyde lyase, 2-hydroxy-1,2-diphenylethanone benzaldehyde-lyase,

Enzyme Mechanism

Introduction

(R)-benzoin is cleaved producing two molecules of benzaldehyde. Thiamine diphosphate (ThDP) is activated by the deprotonation of a carbon and the carbanion reacts with (R)-benzoin producing a tetrahedral intermediate. Benzaldehyde is eliminated leaving an enamine. At low benzaldehyde concentration the enamine is protonated via a water molecule attached to His29 and then the other benzaldehyde molecule is released.

Alternatively, at high benzaldehyde concentration carboligation of the enamine with another benzaldehyde molecule generates (R)-benzoin but this does not occur under normal circumstances.

In fact, alteration of a single residue (A28S) converts benzaldehyde lyase into a benzoylformate decarboxylase.

Catalytic Residues Roles

UniProt PDB* (2ag0)
Gly419 (main-C) Gly419A (main-C) Forms a hydrogen bond to the N1' of the ThDP cofactor and is thought to induce the 1',4'-imino tautomer in the pyrimidine ring. (PMID:16302970) Circular dichroism studies of other thiamin diphosphate (ThDP)-dependent enzymes such as the E1 subunit of Escherichia coli pyruvate dehydrogenase demonstrate the presence of the 1',4'-imino tautomer of ThDP. (PMID:15157089) hydrogen bond acceptor, electrostatic stabiliser
His29 His29B H29 supposedly deprotonates the hydroxyl group on the tetrahedral intermediate resulting in cleavage of the tetrahedral intermediate with the release of the first benzaldehyde molecule (PMID:16302970). It also hydrogen bonds to a water molecule which is thought to protonate the enamine in the subsequent step leading to the release of the second benzaldehyde molecule (PMID:16302970). hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Glu50 Glu50B Forms a hydrogen bond to the N1' on the pyrimidine of ThDP. (PMID:16302970) Activation of ThDP cofactor by Glu50, has been inferred based upon the other thiamine dependent reactions in MACiE. Glu50 is thought to deprotonate N1' leading to the formation of the ThDP carbanion. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Gln113 Gln113B Comparisons of X-ray structures of BAL with BFD suggest that Gln113 may have a role in stabilising the transition state. It also forms hydrogen bonds to His29 and water. 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, proton relay, bimolecular elimination, overall product formed, intermediate collapse, native state of cofactor regenerated, native state of enzyme regenerated

References

  1. Kneen MM et al. (2005), Biochim Biophys Acta, 1753, 263-271. Exploring the active site of benzaldehyde lyase by modeling and mutagenesis. DOI:10.1016/j.bbapap.2005.08.025. PMID:16226928.
  2. Brandt GS et al. (2008), Biochemistry, 47, 7734-7743. Probing the active center of benzaldehyde lyase with substitutions and the pseudosubstrate analogue benzoylphosphonic acid methyl ester. DOI:10.1021/bi8004413. PMID:18570438.
  3. Chakraborty S et al. (2008), Biochemistry, 47, 3800-3809. Mechanism of benzaldehyde lyase studied via thiamin diphosphate-bound intermediates and kinetic isotope effects. DOI:10.1021/bi702302u. PMID:18314961.
  4. Mosbacher TG et al. (2005), FEBS J, 272, 6067-6076. Structure and mechanism of the ThDP-dependent benzaldehyde lyase from Pseudomonas fluorescens. DOI:10.1111/j.1742-4658.2005.04998.x. PMID:16302970.
  5. Nemeria N et al. (2004), Biochemistry, 43, 6565-6575. Tetrahedral Intermediates in Thiamin Diphosphate-Dependent Decarboxylations Exist as a 1‘,4‘-Imino Tautomeric Form of the Coenzyme, Unlike the Michaelis Complex or the Free Coenzyme†. DOI:10.1021/bi049549r. PMID:15157089.

Step 1. Glu50B deprotonates the thiamine diphosphate cofactor, which initiates double bond rearrangement that results in the deprotonation of the N=CH-S group, activating the cofactor.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu50B hydrogen bond acceptor
Gly419A (main-C) hydrogen bond acceptor, electrostatic stabiliser
His29B hydrogen bond donor
Gln113B hydrogen bond acceptor, electrostatic stabiliser
Glu50B 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 benzoin in a nucleophilic addition. Kneen et al. suggest that the protonation event might be mediated through a water molecule and the His29B, with the Gln113B activating the His29B [PMID:16226928].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu50B hydrogen bond donor
Gly419A (main-C) hydrogen bond acceptor, electrostatic stabiliser
His29B hydrogen bond donor
Gln113B hydrogen bond acceptor, electrostatic stabiliser
His29B proton donor

Chemical Components

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

Step 3. His29B deprotonates one of the hydroxyl groups (again, mediated through a water molecule) of the bound intermediate, initiating an elimination that productes one of the benzaldehyde products. Thiamine diphosphate acts as an electron sink.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu50B hydrogen bond donor
Gly419A (main-C) hydrogen bond acceptor, electrostatic stabiliser
His29B hydrogen bond donor
Gln113B hydrogen bond acceptor, electrostatic stabiliser
His29B proton acceptor

Chemical Components

ingold: bimolecular elimination, 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 His29B.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu50B hydrogen bond donor
Gly419A (main-C) hydrogen bond acceptor, electrostatic stabiliser
His29B hydrogen bond donor
Gln113B hydrogen bond acceptor, electrostatic stabiliser
His29B proton donor

Chemical Components

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

Step 5. His29B deprotonates the second hydroxyl group of the intermediate, initiating an elimination that results in a reformation of the carbanionic activated cofactor and the second benzaldehyde product.)Substrates=(M0221-I3,2ag0-His29B

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu50B hydrogen bond donor
Gly419A (main-C) hydrogen bond acceptor, electrostatic stabiliser
His29B hydrogen bond acceptor, hydrogen bond donor
Gln113B hydrogen bond acceptor, electrostatic stabiliser
His29B 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 Glu50B.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu50B hydrogen bond donor
Gly419A (main-C) hydrogen bond acceptor, electrostatic stabiliser
His29B hydrogen bond donor
Gln113B hydrogen bond acceptor, electrostatic stabiliser
Glu50B 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. Glu50B 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 benzoin in a nucleophilic addition. Kneen et al. suggest that the protonation event might be mediated through a water molecule and the His29B, with the Gln113B activating the His29B [PMID:16226928].

Step 3. His29B deprotonates one of the hydroxyl groups (again, mediated through a water molecule) of the bound intermediate, initiating an elimination that productes one of the benzaldehyde products. Thiamine diphosphate acts as an electron sink.

Step 4. Thiamine diphosphate initiates a double bond rearrangement, which results in the intermediate being protonated by His29B.

Step 5. His29B deprotonates the second hydroxyl group of the intermediate, initiating an elimination that results in a reformation of the carbanionic activated cofactor and the second benzaldehyde product.)Substrates=(M0221-I3,2ag0-His29B

Step 6. The carbanion of the thiamine diphosphate cofactor deprotonates the adjacent amine, which initiates double bond rearrangement that results in the deprotonation of Glu50B.

Products.

Contributors

Gemma L. Holliday, James Willey