Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati 517507, India.
Appl Biochem Biotechnol. 2013 Feb;169(3):862-9. doi: 10.1007/s12010-012-0027-8. Epub 2013 Jan 4.
The Krebs cycle dictates oxidative and reductive conditions in Staphylococcus aureus and is mainly regulated by isocitrate dehydrogenase (IDH) which plays pivotal role in the growth and pathogenesis of the bacteria. In the present study, IDH gene from S. aureus ATCC12600 was cloned in the Sma I site of pQE 30 vector; the resultant clone was named as UVIDH1. The insert in the clone was sequenced (accession number HM067707), and the sequence showed complete homology with IDH sequence of other S. aureus strains reported in the database indicating presence of single enzyme in S. aureus, and considerable sequence homology with other bacteria was observed; however, only 24% homology was found with NADP-dependent human IDH. Phylogenetically, the S. aureus IDH showed close identity with Bacillus subtilis and high degree of variability with other bacteria and human IDH. The expression of IDH in the clone UVIDH1 was induced with 1 mM IPTG, and the recombinant IDH was purified by passing through nickel metal chelate column; the purified recombinant IDH showed a single band in SDS-PAGE with a molecular weight of 40 kDa; K(m) and V(max) for isocitrate are 8.2 ± 0.28 and 525 ± 25 μM NADPH/mg/min, respectively, and for cofactor NADP 67.5 ± 2.82 μM and V(max) 50.5 ± 2.12 μM NADPH/mg/min.
克雷布斯循环决定金黄色葡萄球菌的氧化还原条件，主要受异柠檬酸脱氢酶 (IDH) 调控，IDH 在细菌的生长和发病机制中起着关键作用。在本研究中，从金黄色葡萄球菌 ATCC12600 中克隆了 IDH 基因，将其插入 pQE 30 载体的 Sma I 位点，所得克隆命名为 UVIDH1。对克隆中的插入片段进行测序（登录号 HM067707），序列与数据库中报道的其他金黄色葡萄球菌菌株的 IDH 序列完全同源，表明金黄色葡萄球菌中存在单一酶，与其他细菌也有相当大的序列同源性，但与 NADP 依赖性人 IDH 的同源性仅为 24%。系统发育分析表明，金黄色葡萄球菌 IDH 与枯草芽孢杆菌亲缘关系密切，与其他细菌和人 IDH 高度可变。用 1 mM IPTG 诱导克隆 UVIDH1 中 IDH 的表达，并用镍金属螯合柱纯化重组 IDH；SDS-PAGE 显示纯化的重组 IDH 有一条 40 kDa 的单带；异柠檬酸的 K(m)和 V(max)分别为 8.2±0.28 和 525±25 μM NADPH/mg/min，NADP 的 K(m)和 V(max)分别为 67.5±2.82 μM 和 50.5±2.12 μM NADPH/mg/min。
