D-2-Hydroxyglutarate dehydrogenase from Pseudomonas aeruginosa PAO1 (PaD2HGDH) catalyzes the oxidation of D-2-hydroxyglutarate to 2ketoglutarate, which is a necessary step in the serine biosynthetic pathway. The dependence of P. aeruginosa on PaD2HGDH makes the enzyme a potential therapeutic target against P. aeruginosa. In this study, recombinant His-tagged PaD2HGDH 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. PaD2HGDH had a k cat value of 11 s −1 and a K m value of 60 μM with D-2hydroxyglutarate 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 k cat /K m 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. PaD2HGDH shares fully conserved residues with other α-hydroxy acid oxidizing enzymes, and these conserved residues are found in the active site of the PaD2HDGH homology model. An Enzyme Function Initiative-Enzyme Similarity Tool Sequence Similarity Network analysis suggests a functional difference between PaD2HGDH and human D2HGDH, and no relationship with VAO. A phylogenetic tree analysis of PaD2HGDH, VAO, and human D2HGDH establishes genetic diversity among these enzymes.

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