SUBSTITUTION AT RESIDUE-473 CONFERS PROGESTERONE 21-HYDROXYLASE ACTIVITY TO CYTOCHROME-P450-2C2

The carboxyl-terminal 28 amino acids of rabbit cytochrome P450 2C2 are markedly different from those of other rabbit cytochrome P450 2C fami ly members and, substitution of the equivalent amino acids of other cy tochrome P450s can confer novel steroid hydroxylase activity to P450 2 C2 while the normal lauric acid hydroxylase activity is retained. To d etermine the basis for the novel steroid hydroxylase activity, amino a cids of cytochrome P450 2C1 were substituted for those of cytochrome P 450 2C2 and the mutants were expressed in COS-1 cells. There are 13 di fferences between the sequences of cytochrome P450 2C2 and P450 2C1 in this region, including five nonconservative exchanges of charged and uncharged amino acids, However, only substitution of valine for Ser-47 3 increased steroid hydroxylase activity to the maximum level expected in a modified cytochrome P450 2C2, which contained additional substit utions in the 368-388 region to maximize progesterone hydroxylase acti vity. Introduction of this single substitution into cytochrome P450 2C 2 resulted in 21-progesterone hydroxylase activity similar to that res ulting from substitution of all 28 carboxyl-terminal cytochrome P450 2 C1 amino acids. None of the substitutions, with one exception, substan tially affected either lauric acid hydroxylase activity or the amount of immunologically reactive cytochrome P450 that was expressed. A glyc ine substitution for Val-477 reduced activity of both lauric acid hydr oxylase and progesterone hydroxylase and altered the regioselectivity of the hydroxylation for both, Homology modeling of cytochrome P450 2C 2, based on the cytochrome bacterial P450cam sequence, indicated that the side chains of residue 473 and the other five residues previously shown to affect substrate specificity face the substrate pocket. For f our of the six residues, smaller and more hydrophobic residues increas ed progesterone relative to lauric acid hydroxylation.