Type 2 isopentenyl diphosphate isomerase from a thermoacidophilic archaeon Sulfolobus shibatae.
Type 2 isopentenyl diphosphate isomerase from a thermoacidophilic archaeon Sulfolobus shibatae.
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DOI：
10.1111/j.1432-1033.2004.04010.x
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发表时间：
2004-03
期刊：
影响因子：
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通讯作者：
S. Yamashita;H. Hemmi;Yosuke Ikeda;T. Nakayama;T. Nishino
中科院分区：
文献类型：
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作者：
S. Yamashita;H. Hemmi;Yosuke Ikeda;T. Nakayama;T. Nishino
虽然异戊烯基二磷酸-二甲基烯丙基二磷酸异构酶被认为是古生菌所必需的，因为它们使用甲氧戊酸途径，但在任何古生菌中都没有检测到相应的活性。最近在一些细菌菌株中报道了一种新型的酶，它与已知的、研究得很好的类型的酶没有序列相似性。在这项研究中，我们克隆了一种新的细菌异构酶的同源基因，该基因来自一株嗜热嗜酸古细菌柴胡。将该基因在大肠杆菌中进行异源表达，并对重组酶进行了纯化和鉴定。这种耐热的古菌酶是四聚体，需要NAD(P)H和镁离子才能发挥活性，这与它的细菌同源物类似。利用它的同工酶，我们能够证实古生菌的酶严格依赖于FMN。此外，我们提供的证据表明，该酶也具有NADH脱氢酶活性，尽管它催化异构酶反应，而不消耗任何可检测到的NADH。
Although isopentenyl diphosphate-dimethylallyl diphosphate isomerase is thought to be essential for archaea because they use the mevalonate pathway, its corresponding activity has not been detected in any archaea. A novel type of the enzyme, which has no sequence similarity to the known, well-studied type of enzymes, was recently reported in some bacterial strains. In this study, we describe the cloning of a gene of a homologue of the novel bacterial isomerase from a thermoacidophilic archaeon Sulfolobus shibatae. The gene was heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. The thermostable archaeal enzyme is tetrameric, and requires NAD(P)H and Mg2+ for activity, similar to its bacterial homologues. Using its apoenzyme, we were able to confirm that the archaeal enzyme is strictly dependent on FMN. Moreover, we provide evidence to show that the enzyme also has NADH dehydrogenase activity although it catalyzes the isomerase reaction without consuming any detectable amount of NADH.