Department of Chemistry, Georgia State University, Atlanta, Georgia, USA.
Department of Chemistry, Georgia State University, Atlanta, Georgia, USA; Department of Biology, Georgia State University, Atlanta, Georgia, USA; Department of The Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia, USA.
J Biol Chem. 2023 Mar;299(3):103008. doi: 10.1016/j.jbc.2023.103008. Epub 2023 Feb 11.
Pseudomonas aeruginosa PAO1 d-2-hydroxyglutarate (D2HG) dehydrogenase (PaD2HGDH) oxidizes D2HG to 2-ketoglutarate during the vital l-serine biosynthesis and is a potential therapeutic target against P. aeruginosa. PaD2HGDH, which oxidizes d-malate as an alternative substrate, has been demonstrated to be a metallo flavoprotein that requires Zn for activity. However, the role of Zn in the enzyme has not been elucidated, making it difficult to rationalize why nature employs both a redox center and a metal ion for catalysis in PaD2HGDH and other metallo flavoenzymes. In this study, recombinant His-tagged PaD2HGDH was purified to high levels in the presence of Zn or Co to investigate the metal's role in catalysis. We found that the flavin reduction step was reversible and partially rate limiting for the enzyme's turnover at pH 7.4 with either D2HG or d-malate with similar rate constants for both substrates, irrespective of whether Zn or Co was bound to the enzyme. The steady-state pL profiles of the k and k/K values with d-malate demonstrate that Zn mediates the activation of water coordinated to the metal. Our data are consistent with a dual role for the metal, which orients the hydroxy acid substrate in the enzyme's active site and rapidly deprotonates the substrate to yield an alkoxide species for hydride transfer to the flavin. Thus, we propose a catalytic mechanism for PaD2HGDH oxidation that establishes Zn as a cofactor required for substrate orientation and activation during enzymatic turnover.
铜绿假单胞菌 PAO1 的 D-2-羟基戊二酸（D2HG）脱氢酶（PaD2HGDH）在重要的 l-丝氨酸生物合成过程中将 D2HG 氧化为 2-酮戊二酸，是对抗铜绿假单胞菌的潜在治疗靶标。已证明，氧化 d-苹果酸作为替代底物的 PaD2HGDH 是一种金属黄素蛋白，其活性需要 Zn。然而，Zn 在该酶中的作用尚未阐明，这使得难以合理说明为什么自然界在 PaD2HGDH 和其他金属黄素酶中既使用氧化还原中心又使用金属离子进行催化。在这项研究中，在存在 Zn 或 Co 的情况下，重组 His 标记的 PaD2HGDH 被高度纯化，以研究金属在催化中的作用。我们发现黄素还原步骤是可逆的，并且在 pH 7.4 下，对于酶的周转率是部分限速步骤，对于 D2HG 或 d-苹果酸都具有相似的速率常数，而不管酶是否与 Zn 或 Co 结合。用 d-苹果酸测定的 k 和 k/K 值的稳态 pL 曲线表明，Zn 介导与金属配位的水的激活。我们的数据与金属的双重作用一致，该作用使羟基酸底物在酶的活性位点中定向，并迅速使底物去质子化，以生成用于向黄素转移氢化物的烷氧基物种。因此，我们提出了 PaD2HGDH 氧化的催化机制，该机制确立了 Zn 作为酶周转过程中底物定向和激活所需的辅因子。
