Monomeric isocitrate dehydrogenase (IDH) stands for a separated subgroup among IDH protein family. Up to now, all reported monomeric IDHs are from prokaryotes. Here, a monomeric IDH from a marine methanogenic archaeon Methanococcoides methylutens (MmIDH) was reported for the first time. BLAST search demonstrated that only a few marine archaea encode the monomeric IDH and all these organisms are methylotrophic. MmIDH shows the highest homology (~ 70%) to the monomeric IDHs from some marine bacteria, suggesting a lateral gene transfer event between marine bacteria and archaea. The monomeric state of MmIDH was determined by size exclusion chromatography. MmIDH is divalent cation-dependent and Mn²⁺ is the most favored. Kinetic analysis showed that MmIDH is highly specific to NADP⁺ and cannot utilize the NAD⁺. The optimal temperature for MmIDH activity is 50 °C and the optimal pH is 8.2. Heat inactivation assay revealed that MmIDH is a mesophilic enzyme. It sustained 50% activity after incubation at 39 °C for 20 min. Moreover, the putative coenzyme binding residues (His590, Arg601, and Arg650) of MmIDH were explored by mutagenesis. The triple mutant H590L/R601D/R650S displayed a 5.93-fold preference for NAD⁺ over NADP⁺, indicating that the coenzyme specificity of MmIDH was significantly switched from NADP⁺ to NAD⁺ by three key mutations.

中文翻译：

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来自海洋产甲烷古生甲烷甲烷球菌甲基尿素的单体异柠檬酸脱氢酶的生化和系统发育特征。

单体异柠檬酸脱氢酶（IDH）代表IDH蛋白家族中一个分离的亚组。到目前为止，所有报道的单体IDH均来自原核生物。在此，首次报道了来自海洋产甲烷的古甲烷甲烷菌（Mthanococcoidesmethylutens）（MmIDH）的单体IDH 。BLAST搜索表明，只有少数海洋古细菌编码单体IDH，并且所有这些生物都是甲基营养型的。MmIDH与某些海洋细菌的单体IDH具有最高的同源性（〜70％），这表明海洋细菌与古细菌之间存在侧向基因转移事件。MmIDH的单体状态通过尺寸排阻色谱法确定。MmIDH是二价阳离子依赖性的，而Mn ²⁺是最优选的。动力学分析表明MmIDH对NADP具有高度特异性⁺并且不能使用NAD ⁺。MmIDH活性的最适温度为50°C，最适pH为8.2。热灭活测定显示MmIDH是嗜温酶。在39°C下孵育20分钟后，它保持50％的活性。此外，通过诱变探索了MmIDH的假定的辅酶结合残基（His590，Arg601和Arg650）。三重突变体H590L / R601D / R650S对NAD ^+的偏好是对NADP ⁺的5.93倍，这表明MmIDH的辅酶特异性通过三个关键突变从NADP ⁺明显转变为NAD ⁺。