Muñoz-Clares Rosario A, González-Segura Lilian, Murillo-Melo Dario S, Riveros-Rosas Héctor
Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad de México, 04510, Mexico.
Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad de México, 04510, Mexico.
Chem Biol Interact. 2017 Oct 1;276:52-64. doi: 10.1016/j.cbi.2017.02.007. Epub 2017 Feb 17.
The catalytic mechanism of the NAD(P)-dependent aldehyde dehydrogenases (ALDHs) involves the nucleophilic attack of the essential cysteine (Cys302, mature HsALDH2 numbering) on the aldehyde substrate. Although oxidation of Cys302 will inactivate these enzymes, it is not yet well understood how this oxidation is prevented. In this work we explore possible mechanisms of protection by systematically analyzing the reported three-dimensional structures and amino acid sequences of the enzymes of the ALDH superfamily. Specifically, we considered the Cys302 conformational space, the structure and residues conservation of the catalytic loop where Cys302 is located, the observed oxidation states of Cys302, the ability of physiological reductants to revert its oxidation, and the presence of vicinal Cys in the catalytic loop. Our analyses suggested that: 1) In the apo-enzyme, the thiol group of Cys302 is quite resistant to oxidation by ambient O or mild oxidative conditions, because the protein environment promotes its high pK. 2) NAD(P) bound in the "hydride transfer" conformation afforded total protection against Cys302 oxidation by an unknown mechanism. 3) If formed, the Cys302-sulfenic acid is protected against irreversible oxidation. 4) Of the physiological reductant agents, the dithiol lipoic acid could reduce a sulfenic or a disulfide bond in the ALDHs active site; glutathione cannot because its thiol group cannot reach Cys302, and other physiological monothiols may be ineffective in those ALDHs where their active site cannot sterically accommodate two molecules of the monothiols. 5) Formation of the disulfides Cys301-Cys302, Cys302-Cys304, Cys302-Cys305 and Cys-302-Cys306 in those ALDHs that have these Cys residues is not probable, because of the permitted Cys conformers as well as the conserved structure and low flexibility of the catalytic loop. 6) Only in some ALDH2, ALDH9, ALDH16 and ALDH23 enzymes, Cys303, alone or in conjunction with Cys301, allows disulfide formation. Interestingly, several of these enzymes are mitochondrial.
NAD(P) 依赖的醛脱氢酶（ALDHs）的催化机制涉及必需半胱氨酸（成熟人源 ALDH2 编号为 Cys302）对醛底物的亲核攻击。虽然 Cys302 的氧化会使这些酶失活，但目前对于如何防止这种氧化还了解甚少。在这项工作中，我们通过系统分析 ALDH 超家族酶的已报道三维结构和氨基酸序列，探索可能的保护机制。具体而言，我们考虑了 Cys302 的构象空间、Cys302 所在催化环的结构和残基保守性、观察到的 Cys302 的氧化态、生理还原剂还原其氧化态以及催化环中邻近半胱氨酸的存在情况。我们的分析表明：1）在脱辅酶中，Cys302 的巯基对环境中的氧气或温和氧化条件具有相当的抗氧化性，因为蛋白质环境提高了其 pK 值。2）以“氢化物转移”构象结合的 NAD(P) 通过未知机制提供了对 Cys302 氧化的完全保护。3）如果形成，Cys302 - 亚磺酸可防止不可逆氧化。4）在生理还原剂中，二硫辛酸可以还原 ALDHs 活性位点中的亚磺酸或二硫键；谷胱甘肽则不能，因为其巯基无法接触到 Cys302，并且在那些活性位点在空间上无法容纳两个单硫醇分子的 ALDHs 中，其他生理单硫醇可能无效。5）在具有这些半胱氨酸残基的 ALDHs 中，不太可能形成 Cys301 - Cys302、Cys302 - Cys304、Cys302 - Cys305 和 Cys - 302 - Cys306 二硫键，这是由于允许的半胱氨酸构象以及催化环保守的结构和低灵活性。6）仅在一些 ALDH2、ALDH9、ALDH16 和 ALDH23 酶中，单独的 Cys303 或与 Cys301 一起可形成二硫键。有趣的是，这些酶中的几种是线粒体酶。
