Abstract
D-2-hydroxyglutarate dehydrogenase from Pseudomonas aeruginosa PAO1 (EC:1.1.99.39; UniProt ID: Q9I6H4) is an FAD- and Zn(2+)-dependent metallo-flavoenzyme (E(FAD)-Zn(2+)) that catalyzes D-2-hydroxyglutarate oxidation to 2-ketoglutarate, playing a crucial role in l-serine biosynthesis and serving as a potential therapeutic target against the bacterium. The enzyme is also active with the alternative metal ion Ni(2+). However, the effects of substituting Zn(2+) with the more electronegative and smaller ionic radius Ni(2+) (E(FAD)-Ni(2+)) on enzyme function remain unexplored. This study utilized steady-state kinetics, rapid reaction kinetics, and kinetic solvent viscosity analysis to investigate the effects of metal ion substitution on the catalytic steps of the enzyme. The kinetic solvent viscosity effects on the k (cat) parameter indicate that the flavin reduction and product release limit the overall catalytic turnover of E(FAD)-Zn(2+), with slopes of 0.52. In contrast, E(FAD)-Ni(2+) exhibits no viscosity effects on the k (cat) and k (cat)/K (m) parameters, indicating rapid substrate dissociation and product release and that the turnover is not limited by product release. The first-order rate constant for flavin reduction at a saturating D-malate concentration, k (red), which reports the hydride transfer reaction, was comparable between E(FAD)-Ni(2+) (k (red) = 84 s(-1)) and E(FAD)-Zn(2+) (k (red) = 68 s(-1)). These findings provide mechanistic insight into how replacing Zn(2+) with Ni(2+) alters the rate-limiting steps in D-2-hydroxyglutarate dehydrogenase. The study also highlights how the physicochemical properties of the metal cofactors, such as electronegativity and ionic radius, can influence the catalytic steps of the enzyme including substrate and product release.