Biochemistry. 2020 Dec 29;59(51):4833-4844. doi: 10.1021/acs.biochem.0c00832. Epub 2020 Dec 10.
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.
PAO1 的 d-2-羟戊二酸脱氢酶（D2HGDH）催化 d-2-羟戊二酸氧化为 2-酮戊二酸，这是丝氨酸生物合成途径中的必要步骤。对 D2HGDH 的依赖性使该酶成为针对 的潜在治疗靶点。在这项研究中，从先前注释为 FAD 结合 PCMH 型结构域蛋白的基因 PA0317 中表达和纯化了重组 His 标记的 D2HGDH，达到了很高的水平。用磷酸二酯酶处理和质谱分析鉴定了酶的辅因子为 FAD，荧光发射。在 pH 7.4 和 25°C 下，D2HGDH 以 11 s 的 值和 60 μM 的 值与 d-2-羟戊二酸反应。该酶也与 d-苹果酸反应，但不与分子氧反应。用 d-苹果酸和吩嗪甲硫酸酯作为电子受体进行稳态动力学研究，建立了一种与 pH 7.4 时乒乓双酶稳态动力学一致的机制。与 d-2-羟戊二酸和 d-苹果酸的 / 值比较表明，d-2-羟戊二酸的 C5 羧酸盐对于酶的底物特异性很重要。该酶的其他同工酶以前被归类为 flavoproteins 的 VAO/PMCH 家族。D2HGDH 与其他 α-羟基酸氧化酶完全保守的残基，这些保守残基存在于 D2HDGH 同源模型的活性位点中。酶功能倡议-酶相似性工具序列相似性网络分析表明 D2HGDH 和人 D2HGDH 之间存在功能差异，与 VAO 没有关系。D2HGDH、VAO 和人 D2HGDH 的系统发育树分析确立了这些酶之间的遗传多样性。
