Dataset Information

Structures of the G81A mutant form of the active chimera of (S)-mandelate dehydrogenase and its complex with two of its substrates.

ABSTRACT: (S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida, a membrane-associated flavoenzyme, catalyzes the oxidation of (S)-mandelate to benzoylformate. Previously, the structure of a catalytically similar chimera, MDH-GOX2, rendered soluble by the replacement of its membrane-binding segment with the corresponding segment of glycolate oxidase (GOX), was determined and found to be highly similar to that of GOX except within the substituted segments. Subsequent attempts to cocrystallize MDH-GOX2 with substrate proved unsuccessful. However, the G81A mutants of MDH and of MDH-GOX2 displayed approximately 100-fold lower reactivity with substrate and a modestly higher reactivity towards molecular oxygen. In order to understand the effect of the mutation and to identify the mode of substrate binding in MDH-GOX2, a crystallographic investigation of the G81A mutant of the MDH-GOX2 enzyme was initiated. The structures of ligand-free G81A mutant MDH-GOX2 and of its complexes with the substrates 2-hydroxyoctanoate and 2-hydroxy-3-indolelactate were determined at 1.6, 2.5 and 2.2 A resolution, respectively. In the ligand-free G81A mutant protein, a sulfate anion previously found at the active site is displaced by the alanine side chain introduced by the mutation. 2-Hydroxyoctanoate binds in an apparently productive mode for subsequent reaction, while 2-hydroxy-3-indolelactate is bound to the enzyme in an apparently unproductive mode. The results of this investigation suggest that a lowering of the polarity of the flavin environment resulting from the displacement of nearby water molecules caused by the glycine-to-alanine mutation may account for the lowered catalytic activity of the mutant enzyme, which is consistent with the 30 mV lower flavin redox potential. Furthermore, the altered binding mode of the indolelactate substrate may account for its reduced activity compared with octanoate, as observed in the crystalline state.

SUBMITTER: Sukumar N

PROVIDER: S-EPMC2685731 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Structures of the G81A mutant form of the active chimera of (S)-mandelate dehydrogenase and its complex with two of its substrates.

Sukumar Narayanasami N Dewanti Asteriani A Merli Angelo A Rossi Gian Luigi GL Mitra Bharati B Mathews F Scott FS

Acta crystallographica. Section D, Biological crystallography 20090515 Pt 6

(S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida, a membrane-associated flavoenzyme, catalyzes the oxidation of (S)-mandelate to benzoylformate. Previously, the structure of a catalytically similar chimera, MDH-GOX2, rendered soluble by the replacement of its membrane-binding segment with the corresponding segment of glycolate oxidase (GOX), was determined and found to be highly similar to that of GOX except within the substituted segments. Subsequent attempts to cocrystallize MDH-GOX2 ...[more]

PMID: 19465768

Similar Datasets

Project description:D(--)-Mandelate dehydrogenase, the first enzyme of the mandelate pathway in the yeast Rhodotorula graminis, catalyses the NAD(+)-dependent oxidation of D(--)-mandelate to phenylglyoxylate. D(--)-2-(Bromoethanoyloxy)-2-phenylethanoic acid ['D(--)-bromoacetylmandelic acid'], an analogue of the natural substrate, was synthesized as a probe for reactive and accessible nucleophilic groups within the active site of the enzyme. D(--)-Mandelate dehydrogenase was inactivated by D(--)-bromoacetylmandelate in a psuedo-first-order process. D(--)-Mandelate protected against inactivation, suggesting that the residue that reacts with the inhibitor is located at or near the active site. Complete inactivation of the enzyme resulted in the incorporation of approx. 1 mol of label/mol of enzyme subunit. D(--)-Mandelate dehydrogenase that had been inactivated with 14C-labelled D(--)-bromoacetylmandelate was digested with trypsin; there was substantial incorporation of 14C into two tryptic-digest peptides, and this was lowered in the presence of substrate. One of the tryptic peptides had the sequence Val-Xaa-Leu-Glu-Ile-Gly-Lys, with the residue at the second position being the site of radiolabel incorporation. The complete sequence of the second peptide was not determined, but it was probably an N-terminally extended version of the first peptide. High-voltage electrophoresis of the products of hydrolysis of modified protein showed that the major peak of radioactivity co-migrated with N tau-carboxymethylhistidine, indicating that a histidine residue at the active site of the enzyme is the most likely nucleophile with which D(--)-bromoacetylmandelate reacts. D(--)-Mandelate dehydrogenase was incubated with phenylglyoxylate and either (4S)-[4-3H]NADH or (4R)-[4-3H]NADH and then the resulting D(--)-mandelate and NAD+ were isolated. The enzyme transferred the pro-R-hydrogen atom from NADH during the reduction of phenylglyoxylate. The results are discussed with particular reference to the possibility that this enzyme evolved by the recruitment of a 2-hydroxy acid dehydrogenase from another metabolic pathway.

Dataset Information

Structures of the G81A mutant form of the active chimera of (S)-mandelate dehydrogenase and its complex with two of its substrates.

Publications

Structures of the G81A mutant form of the active chimera of (S)-mandelate dehydrogenase and its complex with two of its substrates.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets