Purification, characterization, and gene analysis of catechol 2,3-dioxygenase from the aniline-assimilating bacterium Pseudomonas species AW-2
Purification, characterization, and gene analysis of catechol 2,3-dioxygenase from the aniline-assimilating bacterium Pseudomonas species AW-2
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DOI：
10.1271/bbb.62.747
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发表时间：
1998-04-01
影响因子：
1.6
通讯作者：
Aoki, K
中科院分区：
文献类型：
--
作者：
Murakami, S;Nakanishi, Y;Aoki, K
从在苯胺上生长的假单胞菌AW-2的细胞提取物中将邻苯二酚2，3-双加氧酶（C23 D; EC 1.13.1.2）纯化至均一，并对纯化的C23 D进行表征。通过凝胶过滤估计的分子量为110 kDa。该酶解离成四个相同的亚基，每个亚基的分子量为33 kDa。该酶对3-甲基儿茶酚和儿茶酚具有高活性，并且不同于来自携带TOL质粒的恶臭假单胞菌mt-2的酶，其对儿茶酚、对3-甲基儿茶酚和4-甲基儿茶酚的外二醇裂解活性以及免疫化学性质最佳。从假单胞菌AW-2的C2, 3D基因alnE推导的氨基酸序列与甲苯胺同化恶臭假单胞菌UCC 22的3-甲基儿茶酚2，3-双加氧酶的氨基酸序列有85.7%的同源性。AlnE与恶臭假单胞菌mt-2中xylE编码的C23 D具有44.1%的同一性。由于XylE对3-甲基儿茶酚具有低活性，因此这些结果表明C23 D之间对3-甲基儿茶酚的底物特异性的差异反映了它们的序列相似性。
Catechol 2,3-dioxygenase (C23D; EC 1.13.1.2) was purified to homogeneity from a cell extract of Pseudomonas sp. AW-2 grown on aniline, and the purified C23D was characterized. The molecular mass estimated by gel filtration was 110 kDa. The enzyme dissociated into four identical subunits each with the molecular mass of 33 kDa. The enzyme had high activity for 3-methylcatechol as well as catechol, and differed from the enzyme from Pseudomonas putida mt-2, which carries the TOL plasmid, in optimal on for catechol, extradiol cleavage activities for 3-methylcatechol and 4-methylcatechol, and immunochemical properties. The amino acid sequence deduced from a C23D gene, alnE, from Pseudomonas sp. AW-2 was 85.7% identical to that of 3-methylcatechol 2,3-dioxygenase from toluidine-assimilating Pseudomonas putida UCC22. AlnE was 44.1% identical to the C23D encoded by xylE in P. putida mt-2. Because XylE has low activity for 3-methylcatechol, these results suggest that the differences in substrate specificity for 3-methylcatechol among the C23Ds reflected their sequence similarity.