Abstract Summary/Description
Pseudomonas aeruginosa is a pathogenic bacterium causing human infections. The bacterium relies heavily on D-2-hydroxyglutarate dehydrogenase for L-serine biosynthesis, making the enzyme a potential target for antibacterial strategies. PaD2HGDH, a Zn2+ and FAD-dependent enzyme, catalyzes the oxidation of D-2-hydroxyglutarate or D-malate to 2-ketoglutarate or oxaloacetate. Metal substitutions of Zn2+ with Cd2+, Co2+, Mn2+, and Ni2+ have been proven to maintain the enzyme's activity, but their effects on the kinetic mechanism of the enzyme are still unclear. In this study, recombinant His-tag bacterial enzyme was purified to high levels with Cd2+, Co2+, Mn2+, Ni2+, and Zn2+ to investigate the effects of the various metals on enzyme catalysis. Irrespective of the metal bound to the bacterial enzyme, 1 mM phenazine methosulfate (PMS), the artificial electron acceptor used in the enzyme kinetic assays, was saturating for all the metal-loaded enzymes with D-malate as substrate. Viscosity studies with glucose were conducted to understand the effects of the various metals on the bacterial enzyme rate-limiting steps at pH 7.4 and 25°C. The viscosity effects on the kcat parameter showed that while the turnover rates of the Co2+, Mn2+, and Zn2+ loaded enzymes were limited by both catalysis and diffusional steps, with slopes of 0.13, 0.47, and 0.52, respectively, the Cd2+ (slope of 1) and Ni2+ (slope of 0) loaded enzymes were fully limited by diffusion and catalysis, respectively. Similar results were obtained when the viscosity effects on the kcat/Km parameter were investigated. The data demonstrate that loading the enzyme with various metals alters the rate-limiting steps during enzyme catalysis with D-malate.
