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

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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.