Genetic and enzymatic analyses of C-P bond cleavage enzyme in bacteria

直接的碳-磷(C-P)键高度稳定，耐化学水解、热分解和光解。因此，在自然生境中，C-P键的生物断裂作为防止磷酸盐积累的一种手段具有重要意义。众所周知，一些细菌细胞使用不同的磷酸盐进行生长。然而，所有检测无细胞系统中C-P键裂解活性的尝试都以失败告终，这些细菌如何以及以什么方式裂解磷酸盐中的C-P键的问题仍未解决。我们首次成功地检测到产气肠杆菌的无细胞提取物中的C-P键裂解活性，并分离出了一种负责该活性的酶。该酶催化C-P键断裂，生成无机磷。没有观察到烷烃的形成。然后将该酶命名为C-P水解酶，以区别于C-P裂解酶和磷酸酶。该酶需要两种蛋白质(E2和E3)才能发挥活性。E_2分子大小为650 kDa。E2的作用是将稳定的C-P键转化为不稳定的C-P键。E3是一个分子量为55 kDa的亚基的同源二聚体。该蛋白的功能是对被E2蛋白不稳定的C-P键进行水解。从该生物体中克隆了覆盖E2和E3结构区的完整基因，并对其结构进行了分析。

A direct carbon-phosphorus (C-P) bond is highly stable and resistant to chemical hydrolysis, thermal decomposition and photolysis. Therefore, biological cleavage of the C-P bond in natural habitats assumes importance as a mean to prevent the accumulation of phosphonates. Some bacterial cells are known to use various phosphonates for growth. However, all attempts to detect C-P bond cleavage activity in cell-free systems have resulted in failure, and the problem of how and by what means these bacteria can cleave the C-P bond in phosphonates remains unsolved. We have succeeded, for the first time, in the detection of C-P bond cleavage activity in cell-free extracts of a bacterium Enterobacter aerogenes, and isolated an enzyme responsible for the activity. The enzyme catalyzed the C-P bond cleavage and produced inorganic phosphate. Formation of alkane was not observed. The enzyme was then designated C-P hydrolase to descriminate from C-P lyase and phosphonatase. The enzyme required two proteins (E2 and E3) for activity. E2 was 650 kDa in molecular size. The function of E2 is to convert stable C-P bond into unstable one. E3 was a homodimer of a subunit with M.W. of 55 kDa. The function of the protein is to hydrolyze C-P bond being unstabled by E2 protein. The entire gene covering E2 and E3 structural regions was cloned from the organism and its structure was also analyzed.