Electrostatic Contribution of K339 to the Flavin Reduction of Pseudomonas aeruginosa PAO1 d -2-Hydroxyglutarate Dehydrogenase

Abstract d-2-Hydroxyglutarate dehydrogenase from Pseudomonas aeruginosa PAO1 (PaD2HGDH; EC 1.1.99.39; UniProt ID Q9I6H4) is a Zn2+- and FAD-dependent enzyme that catalyzes the oxidation of d-2-hydroxyglutarate to 2-ketoglutarate and has emerged as a potential antibacterial target. A fully conserved positive charge, K339 in PaD2HGDH, is located at the active-site entrance ∼2 Å from the substrate C5 carboxylate, where its positively charged side chain is poised to assist substrate binding via electrostatic interactions. To explore its mechanistic significance, K339 was substituted with methionine, glutamine, or arginine. Rapid-reaction kinetics revealed that K339 substitution weakened d-malate binding (Kd) by ∼5 kJ/mol, reflecting loss or partial attenuation of the electrostatic stabilization provided by the lysine side chain to the substrate C5 carboxylate. The rapid-reaction kinetics pH profile showed that loss of the K339 positive charge shifted the pKa of the Zn2+–OH(C2) substrate complex from 8.9 to 10.4, indicating less favorable deprotonation of the substrate C2 hydroxyl in K339 variants. The pH-independent rate constant for flavin reduction, kred, decreased by up to 80-fold (ΔΔG ∼ 11 kJ/mol) in K339 variants, consistent with the mutation altering the optimal orientation of the substrate C2 hydroxyl group required for flavin reduction. All K339 variants markedly impaired kcat and kcat/Km, revealing a ∼15 kJ/mol catalytic contribution, while viscosity effects indicated mutation-dependent perturbation of internal isomerization of the enzyme complex during turnover. Overall, K339 plays key electrostatic role in substrate binding and catalysis in PaD2HGDH, with important implications for enzyme engineering and the development of antibacterial inhibitors against P. aeruginosa.