Institute of Molecular Biology and Biotechnology and the Research Center of Life Omics and Health, College of Life Sciences, Anhui Normal University, Wuhu, 241000, Anhui, China.
Institute of Molecular Biology and Biotechnology and the Research Center of Life Omics and Health, College of Life Sciences, Anhui Normal University, Wuhu, 241000, Anhui, China.
Biochimie. 2019 May;160:148-155. doi: 10.1016/j.biochi.2019.03.007. Epub 2019 Mar 12.
Monomeric isocitrate dehydrogenases (IDHs) have once been proposed to be exclusively NADP-specific. Intriguingly, we recently have reported an NAD-specific monomeric IDH from Campylobacter sp. FOBRC14 (CaIDH). Moreover, bioinformatic analysis revealed at least three different coenzyme-binding motifs among Campylobacter IDHs. Besides the NAD-binding motif in CaIDH (Leu/Asp/Ser), a typical NADP-binding motif was also identified in Campylobacter corcagiensis IDH (CcoIDH, His/Arg/Arg). Meanwhile, a third putative NAD-binding motif was found in Campylobacter concisus IDH (CcIDH, Leu/Leu/Ala). In this study, CcIDH was overexpressed in Escherichia coli and purified to homogeneity. Gel filtration chromatography demonstrated that the recombinant CcIDH exists as a monomer in solution. Kinetic analysis showed that the K value of CcIDH for NADP was over 49-fold higher than that for NAD and the catalytic efficiency (k/K) of CcIDH is 115-fold greater for NAD than NADP. Thus, CcIDH is indeed an NAD-specific enzyme. However, the catalytic efficiency (k/K = 0.886 μM s) of CcIDH for NAD is much lower (<5%) when compared to that of the typical monomeric NADP-IDHs for NADP. Then, the three core NAD-binding sites were evaluated by site-directed mutagenesis. The mutant CcIDH (HRR) showed a 51-fold higher K value for NAD and 21-fold lower K value for NADP as compared to that of the wild type enzyme, respectively. The overall specificity of the mutant CcIDH was 12-fold greater for NADP than that for NAD. Thus, the coenzyme specificity of CcIDH was converted from NAD to NADP. Isocitrate dependence of enzyme kinetics showed that although the mutant HRR preferred NADP as its coenzyme, its catalytic efficiency for isocitrate reduced to half of that for the wild-type CcIDH as using NAD. The finding of both NAD and NADP-binding sites in monomeric IDHs from Campylobacter species will provide us a chance to explore the evolution of the coenzyme specificity in monomeric IDH subfamily.
单体异柠檬酸脱氢酶 (IDH) 曾经被认为是专门的 NADP 特异性。有趣的是，我们最近报道了一种来自弯曲杆菌属 FOBRC14 (CaIDH) 的 NAD 特异性单体 IDH。此外，生物信息学分析揭示了弯曲杆菌属 IDH 中至少有三个不同的辅酶结合基序。除了 CaIDH 中的 NAD 结合基序（亮氨酸/天冬氨酸/丝氨酸）外，还在 Campylobacter corcagiensis IDH (CcoIDH，组氨酸/精氨酸/精氨酸) 中鉴定出了典型的 NADP 结合基序。同时，在 Campylobacter concisus IDH (CcIDH，亮氨酸/亮氨酸/丙氨酸) 中发现了第三个可能的 NAD 结合基序。在这项研究中，CcIDH 在大肠杆菌中过表达并纯化至均一性。凝胶过滤层析表明，重组 CcIDH 在溶液中以单体形式存在。动力学分析表明，CcIDH 对 NADP 的 K 值比 NAD 高 49 倍，对 NAD 的催化效率 (k/K) 比 NADP 高 115 倍。因此，CcIDH 确实是一种 NAD 特异性酶。然而，与典型的 NADP-IDHs 相比，CcIDH 对 NAD 的催化效率 (k/K=0.886 μM s) 要低得多（<5%）。然后，通过定点突变评估了三个核心 NAD 结合位点。与野生型酶相比，突变型 CcIDH (HRR) 对 NAD 的 K 值增加了 51 倍，对 NADP 的 K 值降低了 21 倍。突变型 CcIDH 的总体特异性对 NADP 比 NAD 高 12 倍。因此，CcIDH 的辅酶特异性从 NAD 转换为 NADP。酶动力学的异柠檬酸依赖性表明，尽管突变型 HRR 更喜欢 NADP 作为其辅酶，但当使用 NAD 时，其对异柠檬酸的催化效率降低到野生型 CcIDH 的一半。在弯曲杆菌属物种的单体 IDH 中发现 NAD 和 NADP 结合位点，为我们探索单体 IDH 亚家族中辅酶特异性的进化提供了机会。
