Key Laboratory of Molecular Evolution and Biodiversity and Institute of Molecular Biology and Biotechnology, College of Life Sciences, Anhui Normal University, Wuhu 241000, China.
Biochimie. 2009 Nov-Dec;91(11-12):1405-10. doi: 10.1016/j.biochi.2009.07.011. Epub 2009 Jul 23.
Isocitrate dehydrogenase (IDH) is one of the key enzymes in the citric acid cycle, which involves in providing energy and biosynthetic precursors for metabolism. Here, we report for the first time the enzymatic characterization of a monomeric NADP(+)-dependent IDH from Streptomyces lividans TK54 (SlIDH). The icd gene (GenBank database accession number EU661252) encoding IDH was cloned and overexpressed in Escherichia coli. The molecular mass of SlIDH was about 80 kDa, typical of a monomeric NADP-IDH, and showed high amino acid sequence identity with known monomeric IDHs. The optimal activity of the 6His-tagged SlIDH was found at pH values 8.5 (Mn(2+)) and 9.0 (Mg(2+)), and the optimal temperature was around 46 degrees C. Heat-inactivation studies showed that about 50% SlIDH activity was preserved at 38 degrees C after 20 min of incubation. The recombinant SlIDH displayed a 62,000-fold (k(cat)/K(m)) preference for NADP(+) over NAD(+) with Mn(2+), and a 85,000-fold greater specificity for NADP(+) than NAD(+) with Mg(2+). Therefore, SlIDH is a divalent cation-dependent monomeric IDH with remarkably high coenzyme preference for NADP(+).
异柠檬酸脱氢酶（IDH）是柠檬酸循环中的关键酶之一，参与为代谢提供能量和生物合成前体。在这里，我们首次报道了来自 Streptomyces lividans TK54（SlIDH）的单体 NADP(+)依赖型 IDH 的酶学特性。编码 IDH 的 icd 基因（GenBank 数据库登录号 EU661252）在大肠杆菌中克隆和过表达。SlIDH 的分子量约为 80 kDa，典型的单体 NADP-IDH，与已知的单体 IDH 具有高度的氨基酸序列同一性。6His 标记的 SlIDH 的最佳活性在 pH 值 8.5（Mn(2+)）和 9.0（Mg(2+)）下发现，最佳温度约为 46 摄氏度。热失活动力学研究表明，在 38 摄氏度孵育 20 分钟后，约有 50%的 SlIDH 活性得以保留。重组 SlIDH 显示出对 Mn(2+)的 NADP(+)的 62,000 倍（k(cat)/K(m)）偏好，以及对 Mg(2+)的 NADP(+)的 85,000 倍更高的特异性。因此，SlIDH 是一种依赖二价阳离子的单体 IDH，对 NADP(+)具有极高的辅酶偏好性。
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