Active-site Mutants of Benzoylformate Decarboxylase Provide a Variety of Implications for the Roles of the Active-site Residues, His70 and His281

Indianapolis, IN

Benzoylformate decarboxylase (BFDC) is a thiamin diphosphate (ThDP)-dependent enzyme, initially isolated from Pseudomonas putida. BFDC catalyzes the decarboxylation of benzoylformic acid to benzaldehyde and carbon dioxide. In a side reaction, BFDC also exhibits carboligase activity enabling the formation of stereospecific α-hydroxy ketones that are of industrial interest. The putative mechanism of BFDC in essence involves four intermediate steps. It is known from previous site-directed mutagenesis studies that the active site residues, Ser26, His70 and His281 play important roles in the catalytic mechanism. In this project, we explore the specific contributions of the His70 and His281 residues to the individual catalytic steps using a recently developed NMR spectroscopy technique. Variants containing mutations at His70 and His281 were over-expressed in E. coli and purified using nickel affinity chromatography. The Michaelis-Menten parameters (kcat and Km) were determined for each variant using a NADH-linked coupled assay. Subsequently, NMR coupled with rapid acid-quench was used to determine relative concentrations of reaction intermediates and thereby, rate constants for the intermediate steps. Here we report those results and discuss their implications for the roles of His70 and His281 in the BFDC mechanism. Moreover, the unanticipated role of His70 in stabilizing ThDP binding with BFDC is also discussed.