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.
Int J Mol Sci. 2016 Feb 26;17(3):296. doi: 10.3390/ijms17030296.
Bifidobacterium longum is a very important gram-positive non-pathogenic bacterium in the human gastrointestinal tract for keeping the digestive and immune system healthy. Isocitrate dehydrogenase (IDH) from B. longum (BlIDH), a novel member in Type II subfamily, was overexpressed, purified and biochemically characterized in detail. The active form of BlIDH was an 83-kDa homodimer. Kinetic analysis showed BlIDH was a NADP⁺-dependent IDH (NADP-IDH), with a 567- and 193-fold preference for NADP⁺ over NAD⁺ in the presence of Mg(2+) and Mn(2+), respectively. The maximal activity for BlIDH occurred at 60 °C (with Mn(2+)) and 65 °C (with Mg(2+)), and pH 7.5 (with Mn(2+)) and pH 8.0 (with Mg(2+)). Heat-inactivation profiles revealed that BlIDH retained 50% of maximal activity after incubation at 45 °C for 20 min with either Mn(2+) or Mg(2+). Furthermore, the coenzyme specificity of BlIDH can be completely reversed from NADP⁺ to NAD⁺ by a factor of 2387 by replacing six residues. This current work, the first report on the coenzyme specificity conversion of Type II NADP-IDHs, would provide better insight into the evolution of NADP⁺ use by the IDH family.
长双歧杆菌是人类胃肠道中一种非常重要的革兰氏阳性非致病性细菌，可保持消化系统和免疫系统的健康。本研究从双歧杆菌中过表达、纯化并详细生化表征了新型 II 型亚家族成员异柠檬酸脱氢酶（IDH）。BlIDH 的活性形式为 83kDa 同源二聚体。动力学分析表明，BlIDH 是一种 NADP⁺依赖性 IDH（NADP-IDH），在存在 Mg²⁺和 Mn²⁺的情况下，对 NADP⁺的亲和力分别是 NAD⁺的 567 倍和 193 倍。BlIDH 的最大活性出现在 60°C（Mn²⁺）和 65°C（Mg²⁺），pH7.5（Mn²⁺）和 pH8.0（Mg²⁺）。热失活动力学表明，BlIDH 在 45°C 下用 Mn²⁺或 Mg²⁺孵育 20 分钟后，保留 50%的最大活性。此外，通过替换六个残基，BlIDH 的辅酶特异性可以从 NADP⁺完全反转到 NAD⁺，倍数为 2387。这项工作首次报道了 II 型 NADP-IDHs 的辅酶特异性转换，将为深入了解 IDH 家族对 NADP⁺利用的进化提供更好的认识。
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