Replacing the carboxy-terminal 28 residues of rabbit liver P-450 (laurate (omega-1)-hydroxylase) with those of P-450 (testosterone 16 alpha-hydroxylase) produces a new stereospecific hydroxylase activity

cDNA for chimeric P-450 consisting of the amino-terminal 462 residues of P-450 (laurate (omega-1)-hydroxylase) and the remaining 28 residues of P-450 (testosterone 16 alpha-hydroxylase) was constructed and expressed in yeast cells. The resulting chimera could catalyze laurate (omega-1)-hydroxylation and benzphetamine N-demethylation at much higher rates than the parental P-450s, but exhibited the same specificity towards fatty acid substrates as the wild-type laurate hydroxylase. When testosterone was examined as a substrate, the 16 beta-hydroxylated product, which cannot be formed by either of the parental P-450s, was detected, suggesting that the laurate hydroxylase contains a structure that is capable of binding testosterone at a proper orientation so that it can be hydroxylated at the 16 beta position.