Construction and characterization of hybrid dehydratases between adenosylcobalamin-dependent diol and glycerol dehydratases.
Construction and characterization of hybrid dehydratases between adenosylcobalamin-dependent diol and glycerol dehydratases.
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DOI：
10.3177/jnsv.53.102
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发表时间：
2007-04
影响因子：
1.6
通讯作者：
T. Sakai;A. Yamasaki;Satoshi Toyofuku;Tsuneo Nishiki;Michio Yunoki;Noriaki Komoto;T. Tobimatsu;T. Toraya
中科院分区：
文献类型：
--
作者：
T. Sakai;A. Yamasaki;Satoshi Toyofuku;Tsuneo Nishiki;Michio Yunoki;Noriaki Komoto;T. Tobimatsu;T. Toraya
腺苷钴胺素依赖性二醇脱氢酶和甘油脱氢酶是催化1，2-二醇脱水为相应醛的同功能酶。虽然它们具有不同的代谢作用，但这两种酶都由三种不同的亚基组成，并具有共同的（α-γ）2结构。为了阐明每个亚基的作用，我们通过基因工程技术构建了产酸克雷伯氏菌二醇脱氢酶和肺炎克雷伯氏菌甘油脱氢酶在所有亚基组合中的杂合脱氢酶的表达质粒。所有的杂合酶在大肠杆菌中以高水平产生，但只有两个杂合酶的α亚基从甘油脱氢酶和β亚基从二醇脱氢酶组成的表现出高活性。底物特异性、对甘油灭活的敏感性以及野生型和杂交酶的单价阳离子特异性主要由其α亚基的来源决定。
Adenosylcobalamin-dependent diol dehydratase and glycerol dehydratase are isofunctional enzymes that catalyze the dehydration of 1,2-diols to the corresponding aldehydes. Although they bear different metabolic roles, both enzymes consist of three different subunits and possess a common (alphabetagamma)2 structure. To elucidate the roles of each subunit, we constructed expression plasmids for the hybrid dehydratases between diol dehydratase of Klebsiella oxytoca and glycerol dehydratase of Klebsiella pneumoniae in all the combinations of subunits by gene engineering techniques. All of the hybrid enzymes were produced in Escherichia coli at high levels, but only two hybrid enzymes consisting of the alpha subunit from glycerol dehydratase and the beta subunits from diol dehydratase showed high activity. The substrate specificity, the susceptibility to inactivation by glycerol, and the monovalent cation specificity of the wild type and hybrid enzymes were primarily determined by the origin of their alpha subunits.