Comparison of the 17α-hydroxylase/C17,20-lyase activities of porcine, guinea pig and bovine P450c17 using purified recombinant fusion proteins containing P450c17 linked to NADPH-P450 reductase

The cDNAs for cytochrome P450c17 (P450c17) of three species, pig, guinea pig, and cow, representing three families of mammals (suidae, procaviidae, and bovidae, respectively) were each engineered into an expression plasmid (pCWori+). The P450c17 domain of the coding sequence was connected to a truncated form of rat NADPH-P450 reductase by a linker sequence encoding two amino acids (SerThr). These fusion proteins were expressed in E. coli and purified for use in enzymatic assays to determine similarities and differences in 17α-hydroxylase and lyase activities. The fusion proteins were found to catalyze both the 17α-hydroxylation of progesterone (P4) and pregnenolone (P5) to 17α-hydroxylated P4 and P5 (17α-OH P4 and 17α-OH P5) followed by the C17,20-lyase reaction for the conversion of these C21-17α-hydroxylated steroids to C19-steroids (the C17,20-lyase reaction). These in vitro studies show that (a) porcine P450c17 possesses cytochrome b5 (b5)-stimulated C17,20-lyase activity that converts 17αOH-P4 to and...