Abstract

The carboxyl-terminal 28 amino acids of rabbit cytochrome P450 2C2 are markedly different from those of other rabbit cytochrome P450 2C family members and, substitution of the equivalent amino acids of other cytochrome P450s can confer novel steroid hydroxylase activity to P450 2C2 while the normal lauric acid hydroxylase activity is retained. To determine the basis for the novel steroid hydroxylase activity, amino acids of cytochrome P450 2C1 were substituted for those of cytochrome P450 2C2 and the mutants were expressed in COS-1 cells. There are 13 differences 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-473 increased steroid hydroxylase activity to the maximum level expected in a modified cytochrome P450 2C2, which contained additional substitutions in the 368-388 region to maximize progesterone hydroxylase activity. Introduction of this single substitution into cytochrome P450 2C2 resulted in 21-progesterone hydroxylase activity similar to that resulting from substitution of all 28 carboxyl-terminal cytochrome P450 2C1 amino acids. None of the substitutions, with one exception, substantially affected either lauric acid hydroxylase activity or the amount of immunologically reactive cytochrome P450 that was expressed. A glycine substitution for Val-477 reduced activity of both lauric acid hydroxylase and progesterone hydroxylase and altered the regioselectivity of the hydroxylation for both. Homology modeling of cytochrome P450 2C2, 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 four of the six residues, smaller and more hydrophobic residues increased progesterone relative to lauric acid hydroxylation.