PDBsum entry: 1mcz

Lyase

PDB id: 1mcz

Contents

Protein chains

(+ 10 more) 524 a.a. *

Ligands

TDP ×16

RMN ×16

Metals

_MG ×24

Waters ×1656

* Residue conservation analysis

PDB id:

1mcz

Name:

Lyase

Title:

Benzoylformate decarboxylase from pseudomonas putida complexed with an inhibitor, r-mandelate

Structure:

Benzoylformate decarboxylase. Chain: a, b, c, d, e, f, g, h, i, j, k, l, m, n, o, p. Synonym: bfd, bfdc. Engineered: yes

Source:

Pseudomonas putida. Organism_taxid: 303. Gene: mdlc. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Tetramer (from PQS)

Resolution:

2.80Å R-factor: 0.200 R-free: 0.220

Authors:

E.S.Polovnikova,A.K.Bera,M.S.Hasson

Key ref:

E.S.Polovnikova et al. (2003). Structural and kinetic analysis of catalysis by a thiamin diphosphate-dependent enzyme, benzoylformate decarboxylase. Biochemistry, 42, 1820-1830. PubMed id: 12590569 DOI: 10.1021/bi026490k

Date:

06-Aug-02 Release date: 25-Feb-03

Protein chains

P20906  (MDLC_PSEPU) -  Benzoylformate decarboxylase

Seq: 528 a.a.

Struc: 524 a.a.

Enzyme reactions

• Enzyme class: E.C.4.1.1.7 - Benzoylformate decarboxylase.
• Reaction: Benzoylformate = benzaldehyde + CO₂

Benzoylformate (Bound ligand (Het Group name = RMN) corresponds exactly) = benzaldehyde + CO(2)

• Cofactor: Thiamine diphosphate

Thiamine diphosphate (Bound ligand (Het Group name = TDP) corresponds exactly)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

 Gene Ontology (GO) functional annotation 

• Biological process: aromatic compound catabolic process (2 terms)
• Biochemical function: catalytic activity (8 terms)

reference

DOI no: 10.1021/bi026490k

Biochemistry 42:1820-1830 (2003)

PubMed id: 12590569

Structural and kinetic analysis of catalysis by a thiamin diphosphate-dependent enzyme, benzoylformate decarboxylase.

E.S.Polovnikova, M.J.McLeish, E.A.Sergienko, J.T.Burgner, N.L.Anderson, A.K.Bera, F.Jordan, G.L.Kenyon, M.S.Hasson.

ABSTRACT

Benzoylformate decarboxylase is a member of the family of enzymes that are dependent on the cofactor thiamin diphosphate. A structure of this enzyme binding (R)-mandelate, a competitive inhibitor, suggests that at least two hydrogen bonds are formed between the substrate, benzoylformate, and active site side chains. The first is between the carboxylate group of benzoylformate and the hydroxyl group of S26, and the second is between carbonyl group of the substrate and an imidazole nitrogen of H70. Steady-state kinetic studies indicate that the catalytic parameters are strongly affected in three active site mutants, S26A, H70A, and H281A. The K(m) of S26A was increased most dramatically, 25-fold more than that of the wild-type enzyme, while the K(i) of (R)-mandelate was increased 100-fold, suggesting that the serine hydroxyl is important for substrate binding. The k(cat) of H70A is reduced more than 3 orders of magnitude, strongly implicating this residue in catalysis, and H281 showed significant, but smaller magnitude, effects on both K(m) and k(cat). Stopped-flow experiments using an alternative substrate, p-nitrobenzoylformate, lead to kinetic resolution of the fate of key thiamin diphosphate-bound intermediates. Together, the experimental results suggest the following roles for residues in the active site. The residue H70 is important for the protonation of the 2-alpha-mandelyl-ThDP intermediate, thereby assisting in decarboxylation, and for the deprotonation of the 2-alpha-hydroxybenzyl-ThDP intermediate, aiding product release. H281 is involved in protonation of the enamine. Surprisingly, S26 appears to be involved not only in substrate binding but also in other steps of the reaction.