Octopine dehydrogenase (ODH) from the bay scallop Argopecten irradians catalyzes the stereoselective reductive amination of pyruvate by arginine using the cofactor NADH to produce D-octopine. ODH possesses the ability to join a chiral amino acid with pyruvate to form a stereochemically pure product with two stereocenters. Secondary amines are often important precursors in pharmaceutical production, but are notoriously difficult to synthesize in lab. ODH offers a potential alternative method for the synthesis of specific enantiomers in order to avoid undesired compounds with differences in functionality and possibly toxicity. We used site saturation mutagenesis of six residues lining the binding pocket to modify the protein to accept alternative substrates. A screen was developed to measure the activity of crude preparations of single-substitution variants against a panel of amino acids. Mutants identified thus far showed no improvement on activity against the substrates tested. Ultimately, promising mutants will be purified and tested by initial-rate kinetics. As the kinetic constants of the wild-type enzyme are unknown, a UV spectroscopic assay was used to determine the kinetic parameters for each native substrate at different temperatures. All of the substrates showed unexpected trends in both Km and kcat; while kcat values increased with temperature, the increase in Km was not commensurate. This trend suggests that ODH efficiency may be evolutionarily tuned to temperature, as the organism relies on ODH for glycolysis to continue during escape swimming at a wide range of temperatures (-4 °C to 20 °C).

Expanding the substrate specificity of octopine dehydrogenase from bay scallops

251st National Meeting (2016)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.