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首页> 外文期刊>Journal of bacteriology >Cloning and characterization of the gene encoding 1-cyclohexenylcarbonyl coenzyme A reductase from Streptomyces collinus.
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Cloning and characterization of the gene encoding 1-cyclohexenylcarbonyl coenzyme A reductase from Streptomyces collinus.

机译：克隆和鉴定来自链霉菌的1-环己烯基羰基辅酶A还原酶的基因。

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摘要

We report the cloning of the gene encoding the 1-cyclohexenylcarbonyl coenzyme A reductase (ChcA) of Streptomyces collinus, an enzyme putatively involved in the final reduction step in the formation of the cyclohexyl moiety of ansatrienin from shikimic acid. The cloned gene, with a proposed designation of chcA, encodes an 843-bp open reading frame which predicts a primary translation product of 280 amino acids and a calculated molecular mass of 29.7 kDa. Highly significant sequence similiarity extending along almost the entire length of the protein was observed with members of the short-chain alcohol dehydrogenase superfamily. The S. collinus chcA gene was overexpressed in Escherichia coli by using a bacteriophage T7 transient expression system, and a protein with a specific ChcA activity was detected. The E. coli-produced ChcA protein was purified and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as the enoyl-coenzyme A reductase protein prepared from S. collinus. The enzyme demonstrated the ability to catalyze, in vitro, three of the reductive steps involved in the formation of cyclohexanecarboxylic acid. An S. collinus chcA mutant, constructed by deletion of a genomic region comprising the 5' end of chcA, lost the ChcA activity and the ability to synthesize either cyclohexanecarboxylic acid or ansatrienin. These results suggest that chcA encodes the ChcA that is involved in catalyzing multiple reductive steps in the pathway that provides the cyclohexanecarboxylic acid from shikimic acid.
机译：我们报道了编码链霉菌的1-环己烯基羰基辅酶A还原酶（ChcA）的基因的克隆，该酶被认为参与了sh草酸从sa草酸形成环己基部分的最终还原步骤。拟议的chcA名称的克隆基因编码一个843-bp的开放阅读框，该框预测280个氨基酸的一级翻译产物和29.7 kDa的计算分子量。在短链醇脱氢酶超家族的成员中观察到了几乎沿蛋白质的整个长度延伸的高度相似的序列。通过使用噬菌体T7瞬时表达系统，在大肠杆菌中过表达柯林链球菌chcA基因，并检测到具有特定ChcA活性的蛋白质。纯化了大肠杆菌生产的ChcA蛋白，并显示出与十二烷基硫酸钠制备的烯酰辅酶A还原酶蛋白在十二烷基硫酸钠-聚丙烯酰胺凝胶上的稳态动力学和电泳迁移率相似。该酶显示出在体外催化涉及环己烷羧酸形成的三个还原步骤的能力。通过缺失包含chcA 5'端的基因组区域而构建的Collinus chcA突变体失去了ChcA活性和合成环己烷羧酸或蒽醌的能力。这些结果表明，chcA编码的ChcA参与催化从sh草酸提供环己烷羧酸的途径中的多个还原步骤。

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