Pelletier D A, Harwood C S
Department of Microbiology and Center for Biocatalysis and Bioprocessing, The University of Iowa, Iowa City 52242, USA.
J Bacteriol. 1998 May;180(9):2330-6. doi: 10.1128/JB.180.9.2330-2336.1998.
2-Ketocyclohexanecarboxyl coenzyme A (2-ketochc-CoA) hydrolase has been proposed to catalyze an unusual hydrolytic ring cleavage reaction as the last unique step in the pathway of anaerobic benzoate degradation by bacteria. This enzyme was purified from the phototrophic bacterium Rhodopseudomonas palustris by sequential Q-Sepharose, phenyl-Sepharose, gel filtration, and hydroxyapatite chromatography. The sequence of the 25 N-terminal amino acids of the purified hydrolase was identical to the deduced amino acid sequence of the badI gene, which is located in a cluster of genes involved in anaerobic degradation of aromatic acids. The deduced amino acid sequence of badI indicates that 2-ketochc-CoA hydrolase is a member of the crotonase superfamily of proteins. Purified BadI had a molecular mass of 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a native molecular mass of 134 kDa as determined by gel filtration. This indicates that the native form of the enzyme is a homotetramer. The purified enzyme was insensitive to oxygen and catalyzed the hydration of 2-ketochc-CoA to yield pimelyl-CoA with a specific activity of 9.7 micromol min(-1) mg of protein(-1). Immunoblot analysis using polyclonal antiserum raised against the purified hydrolase showed that the synthesis of BadI is induced by growth on benzoate and other proposed benzoate pathway intermediates but not by growth on pimelate or succinate. An R. palustris mutant, carrying a chromosomal disruption of badI, did not grow with benzoate and other proposed benzoate pathway intermediates but had wild-type doubling times on pimelate and succinate. These data demonstrate that BadI, the 2-ketochc-CoA hydrolase, is essential for anaerobic benzoate metabolism by R. palustris.
2-酮环己烷羧酸辅酶A（2-酮chc-CoA）水解酶被认为可催化一种不寻常的水解环裂解反应，这是细菌厌氧降解苯甲酸途径中最后一个独特步骤。该酶通过依次使用Q-琼脂糖、苯基琼脂糖、凝胶过滤和羟基磷灰石色谱法，从光合细菌沼泽红假单胞菌中纯化得到。纯化后的水解酶25个N端氨基酸序列与badI基因推导的氨基酸序列相同，badI基因位于参与芳香酸厌氧降解的基因簇中。badI推导的氨基酸序列表明，2-酮chc-CoA水解酶是巴豆酸酶超家族蛋白质的成员。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，纯化后的BadI分子量为35 kDa，通过凝胶过滤测定，其天然分子量为134 kDa。这表明该酶的天然形式是同四聚体。纯化后的酶对氧气不敏感，催化2-酮chc-CoA水合生成庚二酰辅酶A，比活性为9.7微摩尔·分钟-1·毫克蛋白质-1。使用针对纯化后的水解酶产生的多克隆抗血清进行免疫印迹分析表明，BadI的合成由在苯甲酸和其他推测的苯甲酸途径中间体上生长诱导，但不由在庚二酸或琥珀酸上生长诱导。携带badI染色体破坏的沼泽红假单胞菌突变体不能利用苯甲酸和其他推测的苯甲酸途径中间体生长，但在庚二酸和琥珀酸上具有野生型倍增时间。这些数据表明，BadI，即2-酮chc-CoA水解酶，对于沼泽红假单胞菌厌氧代谢苯甲酸至关重要。
