Kinetic and Bioinformatic Characterization of d-2-Hydroxyglutarate Dehydrogenase from PAO1.

d-2-Hydroxyglutarate dehydrogenase from PAO1 (D2HGDH) catalyzes the oxidation of d-2-hydroxyglutarate to 2-ketoglutarate, which is a necessary step in the serine biosynthetic pathway. The dependence of on D2HGDH makes the enzyme a potential therapeutic target against . In this study, recombinant His-tagged D2HGDH was expressed and purified to high levels from gene PA0317, which was previously annotated as an FAD-binding PCMH-type domain-containing protein. The enzyme cofactor was identified as FAD with fluorescence emission after phosphodiesterase treatment and with mass spectrometry analysis. D2HGDH had a value of 11 s and a value of 60 μM with d-2-hydroxyglutarate at pH 7.4 and 25 °C. The enzyme was also active with d-malate but did not react with molecular oxygen. Steady-state kinetics with d-malate and phenazine methosulfate as an electron acceptor established a mechanism that was consistent with ping-pong bi-bi steady-state kinetics at pH 7.4. A comparison of the / values with d-2-hydroxyglutarate and d-malate suggested that the C5 carboxylate of d-2-hydroxyglutarate is important for the substrate specificity of the enzyme. Other homologues of the enzyme have been previously grouped in the VAO/PMCH family of flavoproteins. D2HGDH shares fully conserved residues with other α-hydroxy acid oxidizing enzymes, and these conserved residues are found in the active site of the D2HDGH homology model. An Enzyme Function Initiative-Enzyme Similarity Tool Sequence Similarity Network analysis suggests a functional difference between D2HGDH and human D2HGDH, and no relationship with VAO. A phylogenetic tree analysis of D2HGDH, VAO, and human D2HGDH establishes genetic diversity among these enzymes.