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首页> 外文期刊>Journal of bacteriology >Aerobic Activity of Escherichia coliAlcohol Dehydrogenase Is Determined by a Single Amino Acid
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Aerobic Activity of Escherichia coliAlcohol Dehydrogenase Is Determined by a Single Amino Acid

机译：大肠杆菌的需氧活性乙醇脱氢酶由单个氨基酸决定

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Expression of the alcohol dehydrogenase gene, adhE, inEscherichia coli is anaerobically regulated at both the transcriptional and the translational levels. To study the AdhE protein, the adhE + structural gene was cloned into expression vectors under the control of the lacZ andtrp c promoters. Wild-type AdhE protein produced under aerobic conditions from these constructs was inactive. Constitutive mutants (adhC) that produced high levels of AdhE under both aerobic and anaerobic conditions were previously isolated. When only the adhE structural gene from one of the adhC mutants was cloned into expression vectors, highly functional AdhE protein was isolated under both aerobic and anaerobic conditions. Sequence analysis revealed that the adhE gene from the adhC mutant contained two mutations resulting in two amino acid substitutions, Ala267Thr and Glu568Lys. Thus,adhC strains contain a promoter mutation and two mutations in the structural gene. The mutant structural gene fromadhC strains was designated adhE*. Fragment exchange experiments revealed that the substitution responsible for aerobic expression in the adhE* clones is Glu568Lys. Genetic selection and site-directed mutagenesis experiments showed that virtually any amino acid substitution for Glu568 produced AdhE that was active under both aerobic and anaerobic conditions. These findings suggest that adhE expression is also regulated posttranslationally and that strict regulation of alcohol dehydrogenase activity in E. coli is physiologically significant.
机译：厌氧调节酒精脱氢酶基因 adhE 在大肠杆菌中的表达。为了研究AdhE蛋白，在 lacZ 和 trp的控制下，将 adhE + 结构基因克隆到表达载体中。 em> c 启动子。在有氧条件下，由这些构建体产生的野生型AdhE蛋白没有活性。先前已分离出在有氧和厌氧条件下均能产生高水平AdhE的组成型突变体（ adhC ）。当只将其中一个 adhC 突变体的 adhE 结构基因克隆到表达载体中时，在有氧和厌氧条件下均分离出功能强大的AdhE蛋白。序列分析显示，来自 adhC 突变体的 adhE 基因含有两个突变，导致两个氨基酸取代，即Ala267Thr和Glu568Lys。因此， adhC 菌株在结构基因中含有一个启动子突变和两个突变。来自 adhC 菌株的突变结构基因被命名为 adhE *。片段交换实验表明负责 adhE *克隆中有氧表达的取代是Glu568Lys。遗传选择和定点诱变实验表明，实际上，Glu568的任何氨基酸替代均可产生在有氧和厌氧条件下均具有活性的AdhE。这些发现表明 adhE 的表达在翻译后也受到调控，并且严格调控 E中的乙醇脱氢酶活性。大肠杆菌具有重要的生理意义。

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