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

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 aeruginosa d-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.