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首页> 外文期刊>Journal of bacteriology >Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and properties of o-succinylbenzoyl-coenzyme A synthetase from Escherichia coli.
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Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and properties of o-succinylbenzoyl-coenzyme A synthetase from Escherichia coli.

机译：甲萘醌（维生素K2）的生物合成：大肠杆菌中邻琥珀酰苯甲酰辅酶A合成酶的过表达，纯化和性质。

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摘要

The coenzyme A (CoA)- and ATP-dependent conversion of o-succinylbenzoic acid [OSB; 4-(2'-carboxyphenyl)-4-oxobutyric acid], to o-succinylbenzoyl-CoA is carried out by the enzyme o-succinylbenzoyl-CoA synthetase. o-Succinylbenzoyl-CoA is a key intermediate in the biosynthesis of menaquinone (vitamin K2) in both gram-negative and gram-positive bacteria. The enzyme has been overexpressed and purified to homogeneity. The purified enzyme was found to have a native molecular mass of 185 kDa as determined by gel filtration column chromatography on Sephacryl S-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis established a subunit molecular mass of 49 kDa. Thus, the enzyme is a homotetramer. The enzyme showed a pH optimum of 7.5 to 8.0 and a temperature optimum of 30 to 40 degrees C. The Km values for OSB, ATP, and CoA were 16, 73.5, and 360 microM, respectively. Of the various metal ions tested, Mg2+ was found to be the most effective in stimulating the enzyme activity. Studies with substrate analogs showed that neither benzoic acid nor benzoylpropionic acid (succinylbenzene) is a substrate for the enzyme. Thus, it appears that both the benzoyl carboxyl group and the succinyl side chain are required for activation of the aliphatic carboxyl group.
机译：辅酶A（CoA）和ATP依赖性的邻琥珀酰苯甲酸[OSB;通过邻琥珀酰苯甲酰基-CoA合成酶将4-（2'-羧苯基）-4-氧代丁酸]转化为邻琥珀酰苯甲酰基-CoA。邻-琥珀酰苯甲酰-CoA是革兰氏阴性菌和革兰氏阳性菌中甲萘醌（维生素K2）生物合成的关键中间体。该酶已过表达并纯化至均一。通过在Sephacryl S-200上的凝胶过滤柱色谱法测定，发现纯化的酶的天然分子量为185kDa。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳确定的亚单位分子量为49 kDa。因此，该酶是高四聚体。该酶显示最适pH值为7.5至8.0，最适温度为30至40摄氏度。OSB，ATP和CoA的Km值分别为16、73.5和360 microM。在测试的各种金属离子中，发现Mg2 +在刺激酶活性方面最有效。对底物类似物的研究表明，苯甲酸和苯甲酰基丙酸（琥珀酰苯）都不是该酶的底物。因此，似乎脂族羧基的活化既需要苯甲酰基羧基又可以琥珀酰侧链。

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