Metal-Triggered\nFAD Reduction in d‑2-Hydroxyglutarate\nDehydrogenase from Pseudomonas aeruginosa PAO1

Alcohol oxidation is an indispensable chemical reaction\nin biological\nsystems. This process, biologically catalyzed by alcohol dehydrogenases\n(ADHs) and alcohol oxidases (AOXs), follows two distinct chemical\nroutes depending on the cofactor. ADHs have been widely demonstrated\nto require Zn<sup>2+</sup>- and NAD(P)<sup>+</sup>-based cosubstrates.\nExcept for galactose oxidase, AOXs achieve their conversion of alcohols\nto aldehydes or ketones using flavin-based cofactors. The FMN-dependent\nα-hydroxy acid-oxidizing enzymes and the glucose–methanol–choline\n(GMC) superfamily abstract their substrate’s α–OH\nproton using a catalytic histidine, leading to substrate oxidation\nand flavin reduction. However, there is no known alcohol oxidation\nmechanism for enzymes requiring both a flavin and a metal. The Pseudomonas aeruginosa d-2-hydroxyglutarate\ndehydrogenase (<i>Pa</i>D2HGDH) is a recently characterized\nα-hydroxy acid dehydrogenase that converts d-2-hydroxyglutarate\nor d-malate to 2-ketoglutarate or oxaloacetate, respectively. <i>Pa</i>D2HGDH requires FAD and Zn<sup>2+</sup> for catalysis.\nPrevious studies on <i>Pa</i>D2HGDH have identified a highly\nconserved active site histidine residue whose position is topologically\nconserved for catalytic bases in FMN-dependent α-hydroxy acid-oxidizing\nenzymes and the GMC superfamily of oxidoreductases. In this study,\nsolvent isotope effects (SIEs) coupled with pL-rate profiles and a\nviscosity control have been used to probe the role of the Zn<sup>2+</sup> cofactor in the C<sup>2</sup>–OH oxidation of d-malate\nand flavin reduction of <i>Pa</i>D2HGDH. The data revealed\nan inverse solvent equilibrium isotope effect (SEIE) of 0.51 ±\n0.09 consistent with a Zn<sup>2+</sup>-triggered abstraction of the\nsubstrate C<sup>2</sup>–OH proton that initiates d-malate oxidation and flavin reduction. The system provides insights\ninto the role of Zn<sup>2+</sup> in the oxidation mechanism of <i>Pa</i>D2HGDH and, by extension, metallo flavoprotein dehydrogenases.