Metal-Triggered FAD Reduction in d-2-Hydroxyglutarate Dehydrogenase from Pseudomonas aeruginosa PAO1.

Alcohol oxidation is an indispensable chemical reaction in biological systems. This process, biologically catalyzed by alcohol dehydrogenases (ADHs) and alcohol oxidases (AOXs), follows two distinct chemical routes depending on the cofactor. ADHs have been widely demonstrated to require Zn2+- and NAD(P)+-based cosubstrates. Except for galactose oxidase, AOXs achieve their conversion of alcohols to aldehydes or ketones using flavin-based cofactors. The FMN-dependent α-hydroxy acid-oxidizing enzymes and the glucose-methanol-choline (GMC) superfamily abstract their substrate's α-OH proton using a catalytic histidine, leading to substrate oxidation and flavin reduction. However, there is no known alcohol oxidation mechanism for enzymes requiring both a flavin and a metal. The Pseudomonas aeruginosad-2-hydroxyglutarate dehydrogenase (PaD2HGDH) is a recently characterized α-hydroxy acid dehydrogenase that converts d-2-hydroxyglutarate or d-malate to 2-ketoglutarate or oxaloacetate, respectively. PaD2HGDH requires FAD and Zn2+ for catalysis. Previous studies on PaD2HGDH have identified a highly conserved active site histidine residue whose position is topologically conserved for catalytic bases in FMN-dependent α-hydroxy acid-oxidizing enzymes and the GMC superfamily of oxidoreductases. In this study, solvent isotope effects (SIEs) coupled with pL-rate profiles and a viscosity control have been used to probe the role of the Zn2+ cofactor in the C2-OH oxidation of d-malate and flavin reduction of PaD2HGDH. The data revealed an inverse solvent equilibrium isotope effect (SEIE) of 0.51 ± 0.09 consistent with a Zn2+-triggered abstraction of the substrate C2-OH proton that initiates d-malate oxidation and flavin reduction. The system provides insights into the role of Zn2+ in the oxidation mechanism of PaD2HGDH and, by extension, metallo flavoprotein dehydrogenases.