Enzymes are potent catalysts that increase biochemical reaction rates by several orders of magnitude. Flavoproteins are a class of enzymes whose classification relies on their ability to react with molecular oxygen (O2) during catalysis using ionizable active site residues. Pseudomonas aeruginosa D-arginine dehydrogenase (PaDADH) is a flavoprotein that oxidizes D-arginine for P. aeruginosa survival and biofilm formation. The crystal structure of PaDADH reveals the interaction of the glutamate 246 (E246) side chain with the substrate and at least three other active site residues, establishing a hydrogen bond network in the active site. Additionally, E246 likely ionizes to facilitate substrate binding during PaDADH catalysis. This study aimed to investigate how replacing the E246 residue with leucine affects PaDADH catalysis and its ability to react with O2 using steady-state kinetics coupled with pH profile studies. The data reveal a gain of O2 reactivity in the E246L variant, resulting in a reduced flavin semiquinone species and superoxide (O2•-) during substrate oxidation. The O2•- reacts with active site protons, resulting in an observed nonstoichiometric slope of 1.5 in the enzyme\'s log (kcat/Km) pH profile with D-arginine. Adding superoxide dismutase results in an observed correction of the slope to 1.0. This study demonstrates how O2•- can alter the slopes of limbs in the pH profiles of flavin-dependent enzymes and serves as a model for correcting nonstoichiometric slopes in elucidating reaction mechanisms of flavoproteins.

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, kcat, 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 E246 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 E246 mutation to glycine, there was an increased rate of d-arginine turnover kcat from 122 to 500 s-1. Likewise, the kcat 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.