Biochemical characterization of isocitrate dehydrogenase from Methylococcus capsulatus reveals a unique NAD+-dependent homotetrameric enzyme
Biochemical characterization of isocitrate dehydrogenase from Methylococcus capsulatus reveals a unique NAD+-dependent homotetrameric enzyme
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DOI：
10.1007/s00203-006-0200-y
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发表时间：
2007-05-01
影响因子：
2.8
通讯作者：
Steen, Ida Helene
中科院分区：
文献类型：
--
作者：
Stokke, Runar;Madern, Dominique;Steen, Ida Helene
克隆了荚膜甲基球菌（McIDH）异柠檬酸脱氢酶（IDH）基因，并在大肠杆菌中过表达。纯化后的酶是NAD（+）依赖酶，在55-60℃的温度下活性最优，中点熔化温度（T (m)）为70℃。分析超离心（AUC）显示为同四聚体状态，因此McIDH是第一个被表征的同四聚体NAD（+）依赖的IDH。基于McIDH和来自嗜热热菌（Thermus thermophilus, TtHDH）的同四聚体同异柠檬酸脱氢酶（homisisocitrate dehydrogenase， HDH）的结构比对，我们确定了McIDH的扣状结构域可能是四聚体化的位点。此外，McIDH与TtHDH的序列同源性（48%）高于先前鉴定的idh。然而，在多种细菌中发现了与McIDH具有高序列一致性（48-57%）的推定NAD(+)- idh，这表明NAD（+）依赖性idh确实广泛存在于细菌结构域内。包括这些新序列在内的系统发育分析揭示了与真核变构调节的NAD(+)-IDH的密切关系，并且IDH的亚家族III被重新定义为包括细菌NAD(+)-和NADP(+)-依赖的IDH。这种明显的关系表明，编码NAD(+)-IDH的线粒体基因来源于mcidh样idh。
The gene encoding isocitrate dehydrogenase (IDH) of Methylococcus capsulatus (McIDH) was cloned and overexpressed in Escherichia coli. The purified enzyme was NAD(+)-dependent with a thermal optimum for activity at 55-60 degrees C and an apparent midpoint melting temperature (T (m)) of 70 degrees C. Analytical ultracentrifugation (AUC) revealed a homotetrameric state, and McIDH thus represents the first homotetrameric NAD(+)-dependent IDH that has been characterized. Based on a structural alignment of McIDH and homotetrameric homoisocitrate dehydrogenase (HDH) from Thermus thermophilus (TtHDH), we identified the clasp-like domain of McIDH as a likely site for tetramerization. McIDH showed moreover, higher sequence identity (48%) to TtHDH than to previously characterized IDHs. Putative NAD(+)-IDHs with high sequence identity (48-57%) to McIDH were however identified in a variety of bacteria showing that NAD(+)-dependent IDHs are indeed widespread within the domain, Bacteria. Phylogenetic analysis including these new sequences revealed a close relationship with eukaryal allosterically regulated NAD(+)-IDH and the subfamily III of IDH was redefined to include bacterial NAD(+)- and NADP(+)-dependent IDHs. This apparent relationship suggests that the mitochondrial genes encoding NAD(+)-IDH are derived from the McIDH-like IDHs.