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

碳-磷（C-P）直接键高度稳定，耐化学水解、热分解和光解。因此，在自然生境中，C-P键的生物裂解作为防止磷酸盐积累的一种手段具有重要意义。已知一些细菌细胞使用各种磷酸盐来生长。然而，所有在无细胞系统中检测C-P键切割活性的尝试都以失败告终，这些细菌如何以及通过什么方式可以在磷酸盐中切割C-P键的问题仍然没有解决。我们首次成功地检测了产气肠杆菌无细胞提取物的C-P键裂解活性，并分离出一种负责该活性的酶。该酶催化C-P键断裂，生成无机磷酸盐。没有观察到烷烃的形成。该酶被命名为C-P水解酶，以区别于C-P裂解酶和磷酸化酶。这种酶需要两种蛋白质（E2和E3）才能发挥活性。E2分子量为650 kDa。E2的作用是将稳定的C-P键转化为不稳定的键。E3是一个亚基的同二聚体，M.W.为55 kDa。该蛋白的功能是水解E2蛋白不稳定的C-P键。克隆了覆盖E2和E3结构区的整个基因，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.