Tobimatsu T, Azuma M, Matsubara H, Takatori H, Niida T, Nishimoto K, Satoh H, Hayashi R, Toraya T
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-Naka, Okayama 700, Japan.
J Biol Chem. 1996 Sep 13;271(37):22352-7. doi: 10.1074/jbc.271.37.22352.
The gld genes encoding adenosylcobalamin-dependent glycerol dehydrase of Klebsiella pneumoniae were cloned by cross-hybridization with a DNA fragment of Klebsiella oxytoca diol dehydrase genes. Since the Escherichia coli clones isolated did not show appreciable enzyme activity, plasmids for high level expression of cloned genes were constructed. The enzyme expressed in E. coli was indistinguishable from the wild-type glycerol dehydrase of K. pneumoniae by the criteria of polyacrylamide gel electrophoretic, immunochemical, and catalytic properties. It was also shown that the recombinant functional enzyme consists of Mr 61,000, 22,000, and 16, 000 subunits. Sequence analysis of the genes revealed four open reading frames separated by 2-12 bases. The sequential three open reading frames from the first to the third (gldA, gldB, and gldC genes) encoded polypeptides of 555, 194, and 141 amino acid residues with predicted molecular weights of 60,659(alpha), 21,355(beta), and 16,104(gamma), respectively. High level expression of these three genes in E. coli produced more than 14-fold higher level of fully active apoenzyme than that in K. pneumoniae. It was thus concluded that these are the genes encoding the subunits of glycerol dehydrase. The deduced amino acid sequences of the three subunits were 71, 58, and 54% identical with those of the alpha, beta, and gamma subunits of diol dehydrase, respectively, but failed to show any apparent homology with other proteins.
通过与产酸克雷伯菌二醇脱水酶基因的DNA片段进行交叉杂交，克隆了编码肺炎克雷伯菌腺苷钴胺素依赖性甘油脱水酶的gld基因。由于分离得到的大肠杆菌克隆未显示出明显的酶活性，因此构建了用于克隆基因高水平表达的质粒。根据聚丙烯酰胺凝胶电泳、免疫化学和催化特性的标准，在大肠杆菌中表达的酶与肺炎克雷伯菌的野生型甘油脱水酶没有区别。还表明重组功能酶由分子量为61,000、22,000和16,000的亚基组成。基因序列分析揭示了四个开放阅读框，它们之间相隔2 - 12个碱基。从第一个到第三个连续的开放阅读框（gldA、gldB和gldC基因）分别编码555、194和141个氨基酸残基的多肽，预测分子量分别为60,659（α）、21,355（β）和16,104（γ）。这三个基因在大肠杆菌中的高水平表达产生的全活性脱辅基酶水平比肺炎克雷伯菌中的高14倍以上。因此得出结论，这些是编码甘油脱水酶亚基的基因。这三个亚基推导的氨基酸序列分别与二醇脱水酶的α、β和γ亚基的氨基酸序列有71%、58%和54%的同一性，但与其他蛋白质没有明显的同源性。
