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Catalytic Site Atlas Version 2.2.12
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CSA entry for 2ag0
Original Entry
Title:
Lyase
Compound:
Benzaldehyde lyase
Mutant:
No
UniProt/Swiss-Prot:
Q9F4L3-Q9F4L3_PSEFL
EC Class:
4.1.2.38
Other CSA Entries:
Overview of all sites for 2ag0
Homologues of 2ag0
Entries for UniProt/Swiss-Prot: Q9F4L3
Entries for EC: 4.1.2.38
Other Databases:
PDB entry: 2ag0
PDBsum entry: 2ag0
UniProt/Swiss-Prot: Q9F4L3
IntEnz entry: 4.1.2.38
Literature Report:
Introduction:
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.
Mechanism:
(R)-benzoin is cleaved producing two molecules of benzaldehyde.

1)Thiamine diphosphate (ThDP) is activated by the deprotonation of a carbon and the carbanion reacts with (R)-benzoin producing a tetrahedral intermediate.
2)Benzaldehyde is eliminated leaving an enamine.
3)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.

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


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Found by:
Literature reference 

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
GLYA 419 419Sidechain
ElectrostaticCofactor
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)
Evidence from paper Evidence concerns Evidence type
PubMed ID 16302970 Current protein Residue is positioned appropriately (ligand position known)
PubMed ID 15157089 Related protein: A1A7G0 Structural similarity to homologue of known mechanism

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
TPPA 602 0
NucleophileSubstrate
The thiamine diphosphate (ThDP) cofactor is activated by deprotonation by Glu50 resulting in the formation of a carbanion which attacks the substrate producing the tetrahedral intermediate.
Evidence from paper Evidence concerns Evidence type
PubMed ID 16229628 Current protein Ligand is essential for catalysis
PubMed ID 16229628 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
HISB 29 29Sidechain
Acid/baseSubstrate
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).
Evidence from paper Evidence concerns Evidence type
PubMed ID 16302970 Current protein Residue is positioned appropriately (ligand position known)
PubMed ID 16226928 Current protein Mutagenesis of residue

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
GLUB 50 50Sidechain
Acid/baseCofactor
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.
Evidence from paper Evidence concerns Evidence type
PubMed ID 16226928 Current protein Conservation of residue
PubMed ID 16302970 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
GLNB 113 113Sidechain
ElectrostaticTransition state
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.
Evidence from paper Evidence concerns Evidence type
PubMed ID 16226928 Current protein Residue is positioned appropriately (ligand position known)
PubMed ID 16226928 Current protein Structural similarity to homologue of known mechanism
PubMed ID 16226928 Current protein Mutagenesis of residue
Notes:

References:
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