Stereochemistry and Charged State Influence Effector Outcomes of d-2-Hydroxyglutarate Dehydrogenase Ligands

d-2-Hydroxyglutarate dehydrogenase (D2HGDH) has recently received considerable attention due to the involvement of d-2-hydroxyglutarate in various medical conditions. This enzyme has been reported to diverge in substrate scope depending on whether its source is prokaryotic or eukaryotic. The D2HGDH from Pseudomonas aeruginosa, PaD2HGDH, is of particular interest due to its requirement for P. aeruginosa survival via the l-serine biosynthesis pathway and its potential use as a therapeutic target against the bacterium. The enzyme, which is active on d-2-hydroxyglutarate (D2HG) and d-malate, is a Zn²⁺- and FAD-dependent dehydrogenase that employs metal-triggered flavin reduction in its catalytic mechanism. While PaD2HGDH is the most extensively studied D2HGDH homologue, no studies have investigated the ligand-binding modalities in the enzyme, and─for that matter─any D2HGDH homologue. This study investigated the inhibition profiles of PaD2HGDH by various D2HG and d-malate analogues. The study demonstrates that stereochemistry and functional groups at the C2 position of ligands are key determinants of binding to PaD2HGDH. The enzyme recognizes d-isomeric ligands as substrates, with l-isomers acting as reversible inhibitors. Ligand binding requires bidentate coordination with the active site Zn²⁺ cofactor, with longer chain ligands and polar ligands having lower K_is and ΔG^o values due to enhanced interactions with the highly polar active site. Hydrophobic and van der Waals interactions also contribute to ligand binding in PaD2HGDH. The study concludes that PaD2HGDH can be reversibly inhibited, providing a foundation for biochemical studies on PaD2HGDH inhibitors, with direct applications to D2HG biosensor development.