The Research Center of Life Omics and Health and Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, Anhui Normal University, Wuhu, 241000, Anhui, China.
Extremophiles. 2020 Mar;24(2):319-328. doi: 10.1007/s00792-020-01156-2. Epub 2020 Jan 22.
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 Mn 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.
单体异柠檬酸脱氢酶 (IDH) 代表 IDH 蛋白家族中的一个分离亚群。到目前为止，所有报道的单体 IDH 都来自原核生物。本文首次报道了一种来自海洋产甲烷古菌 Methanococcoides methylutens (MmIDH) 的单体 IDH。BLAST 搜索表明，只有少数海洋古菌编码单体 IDH，而所有这些生物都是甲基营养型的。MmIDH 与一些海洋细菌的单体 IDH 具有最高的同源性（~70%），这表明海洋细菌和古菌之间发生了水平基因转移事件。通过尺寸排阻色谱法确定了 MmIDH 的单体状态。MmIDH 是二价阳离子依赖性的，Mn 是最优选的。动力学分析表明，MmIDH 对 NADP 具有高度特异性，不能利用 NAD。MmIDH 活性的最佳温度为 50°C，最佳 pH 值为 8.2。热失活动力学分析表明，MmIDH 是一种嗜中温酶。它在 39°C 孵育 20 分钟后仍能保持 50%的活性。此外，还通过突变研究了 MmIDH 的假定辅酶结合残基（His590、Arg601 和 Arg650）。三重突变体 H590L/R601D/R650S 对 NAD 的偏好性提高了 5.93 倍，表明通过三个关键突变，MmIDH 的辅酶特异性从 NADP 显著切换为 NAD。
