Targeted Mutation of a Non-catalytic Gating Residue Increases the Rate of Pseudomonas aeruginosad-Arginine Dehydrogenase Catalytic Turnover

[Image: see text] Commercial food and l-amino acid industries rely on bioengineered d-amino acid oxidizing enzymes to detect and remove d-amino acid contaminants. However, the bioengineering of enzymes to generate faster biological catalysts has proven difficult as a result of the failure to target specific kinetic steps that limit enzyme turnover, k(cat), and the poor understanding of loop dynamics critical for catalysis. Pseudomonas aeruginosad-arginine dehydrogenase (PaDADH) oxidizes most d-amino acids and is a good candidate for application in the l-amino acid and food industries. The side chain of the loop L2 E(246) residue located at the entrance of the PaDADH active site pocket potentially favors the closed active site conformation and secures the substrate upon binding. This study used site-directed mutagenesis, steady-state, and rapid reaction kinetics to generate the glutamine, glycine, and leucine variants and investigate whether increasing the rate of product release could translate to an increased enzyme turnover rate. Upon E(246) mutation to glycine, there was an increased rate of d-arginine turnover k(cat) from 122 to 500 s(–1). Likewise, the k(cat) values increased 2-fold for the glutamine or leucine variants. Thus, we have engineered a faster biocatalyst for industrial applications by selectively increasing the rate of the PaDADH product release.