Wu Ming-Cai, Tian Chang-Qing, Cheng Hong-Mei, Xu Lei, Wang Peng, Zhu Guo-Ping
Institute of Molecular Biology and Biotechnology, Anhui Normal University, No. 1 Beijing East Road, Wuhu, 241000, Anhui, China; Anhui Province Key Laboratory of Active Biological Macro-molecules, Wannan Medical College, No. 22 Wenchang West Road, Wuhu, 241002, Anhui, China.
Institute of Molecular Biology and Biotechnology, Anhui Normal University, No. 1 Beijing East Road, Wuhu, 241000, Anhui, China.
PLoS One. 2015 May 5;10(5):e0125229. doi: 10.1371/journal.pone.0125229. eCollection 2015.
In most living organisms, isocitrate dehydrogenases (IDHs) convert isocitrate into ɑ-ketoglutarate (ɑ-KG). Phylogenetic analyses divide the IDH protein family into two subgroups: types I and II. Based on cofactor usage, IDHs are either NAD+-specific (NAD-IDH) or NADP+-specific (NADP-IDH); NADP-IDH evolved from NAD-IDH. Type I IDHs include NAD-IDHs and NADP-IDHs; however, no type II NAD-IDHs have been reported to date. This study reports a novel type II NAD-IDH from the marine bacterium Congregibacter litoralis KT71 (ClIDH, GenBank accession no. EAQ96042). His-tagged recombinant ClIDH was produced in Escherichia coli and purified; the recombinant enzyme was NAD+-specific and showed no detectable activity with NADP+. The Km values of the enzyme for NAD+ were 262.6±7.4 μM or 309.1±11.2 μM with Mg2+ or Mn2+ as the divalent cation, respectively. The coenzyme specificity of a ClIDH Asp487Arg/Leu488His mutant was altered, and the preference of the mutant for NADP+ was approximately 24-fold higher than that for NAD+, suggesting that ClIDH is an NAD+-specific ancestral enzyme in the type II IDH subgroup. Gel filtration and analytical ultracentrifugation analyses revealed the homohexameric structure of ClIDH, which is the first IDH hexamer discovered thus far. A 163-amino acid segment of CIIDH is essential to maintain its polymerization structure and activity, as a truncated version lacking this region forms a non-functional monomer. ClIDH was dependent on divalent cations, the most effective being Mn2+. The maximal activity of purified recombinant ClIDH was achieved at 35°C and pH 7.5, and a heat inactivation experiment showed that a 20-min incubation at 33°C caused a 50% loss of ClIDH activity. The discovery of a NAD+-specific, type II IDH fills a gap in the current classification of IDHs, and sheds light on the evolution of type II IDHs.
在大多数生物体内，异柠檬酸脱氢酶（IDHs）将异柠檬酸转化为α-酮戊二酸（α-KG）。系统发育分析将IDH蛋白家族分为两个亚组：I型和II型。根据辅因子的使用情况，IDHs要么是NAD+特异性的（NAD-IDH），要么是NADP+特异性的（NADP-IDH）；NADP-IDH由NAD-IDH进化而来。I型IDHs包括NAD-IDHs和NADP-IDHs；然而，迄今为止尚未报道过II型NAD-IDHs。本研究报道了一种来自海洋细菌滨海聚球藻KT71（ClIDH，GenBank登录号EAQ96042）的新型II型NAD-IDH。带有His标签的重组ClIDH在大肠杆菌中产生并纯化；该重组酶是NAD+特异性的，对NADP+未表现出可检测到的活性。该酶对NAD+的Km值，以Mg2+或Mn2+作为二价阳离子时，分别为262.6±7.4 μM或309.1±11.2 μM。ClIDH Asp487Arg/Leu488His突变体的辅酶特异性发生了改变，该突变体对NADP+的偏好性比对NAD+的偏好性高约24倍，这表明ClIDH是II型IDH亚组中的一种NAD+特异性祖先酶。凝胶过滤和分析超速离心分析揭示了ClIDH的同型六聚体结构，这是迄今为止发现的首个IDH六聚体。CIIDH的一个163个氨基酸的片段对于维持其聚合结构和活性至关重要，因为缺少该区域的截短版本会形成无功能的单体。ClIDH依赖于二价阳离子，最有效的是Mn2+。纯化的重组ClIDH在35°C和pH 7.5时达到最大活性，热失活实验表明，在33°C孵育20分钟会导致ClIDH活性丧失50%。一种NAD+特异性的II型IDH的发现填补了当前IDHs分类中的一个空白，并为II型IDHs的进化提供了线索。
