Tobimatsu T, Hara T, Sakaguchi M, Kishimoto Y, Wada Y, Isoda M, Sakai T, Toraya T
Department of Biotechnology, Faculty of Engineering, Okayama University, Japan.
J Biol Chem. 1995 Mar 31;270(13):7142-8. doi: 10.1074/jbc.270.13.7142.
The pdd genes encoding adenosylcobalamin-dependent diol dehydrase of Klebsiella oxytoca were cloned by using a synthetic oligodeoxyribonucleotide as a hybridization probe followed by measuring the enzyme activity of each clone. Five clones of Escherichia coli exhibited diol dehydrase activity. At least one of them was shown to express diol dehydrase genes under control of their own promoter. Sequence analysis of the DNA fragments found in common in the inserts of these five clones and the flanking regions revealed four open reading frames separated by 10-18 base pairs. The sequential three open reading frames from the second to the fourth (pddA, pddB, and pddC genes) encoded polypeptides of 554, 224, and 173 amino acid residues with predicted molecular weights of 60,348 (alpha), 24,113 (beta), and 19,173 (gamma), respectively. Overexpression of these three genes in E. coli produced more than 50-fold higher level of functional apodiol dehydrase than that in K. oxytoca. The recombinant enzyme was indistinguishable from the wild-type one of K. oxytoca by the criteria of polyacrylamide gel electrophoretic and immunochemical properties. It was thus concluded that these three gene products are the subunits of functional diol dehydrase. Comparisons of the deduced amino acid sequences of the three subunits with other proteins failed to reveal any apparent homology.
通过使用合成的寡聚脱氧核糖核苷酸作为杂交探针，随后测定每个克隆的酶活性，克隆了编码产酸克雷伯菌腺苷钴胺素依赖性二醇脱水酶的pdd基因。五个大肠杆菌克隆表现出二醇脱水酶活性。其中至少一个在其自身启动子的控制下表达二醇脱水酶基因。对这五个克隆的插入片段及其侧翼区域中共同发现的DNA片段进行序列分析，发现四个开放阅读框，它们之间相隔10 - 18个碱基对。从第二个到第四个连续的三个开放阅读框（pddA、pddB和pddC基因）分别编码554、224和173个氨基酸残基的多肽，预测分子量分别为60,348（α）、24,113（β）和19,173（γ）。这三个基因在大肠杆菌中的过表达产生的功能性脱辅基二醇脱水酶水平比产酸克雷伯菌中的高50倍以上。根据聚丙烯酰胺凝胶电泳和免疫化学性质的标准，重组酶与产酸克雷伯菌的野生型酶没有区别。因此得出结论，这三个基因产物是功能性二醇脱水酶的亚基。将这三个亚基的推导氨基酸序列与其他蛋白质进行比较，未发现任何明显的同源性。
