Change in enantioselectivity in bufuralol 1''-hydroxylation by the substitution of phenylalanine-120 by alanine in cytochrome P450 2D6.

The functional roles of phenylalanine at position 120 in drug oxidation by cytochrome P450 2D6 (CYP2D6) were examined using a yeast cell expression system and bufuralol (BF) enantiomers as a chiral substrate. Two mutated cDNAs, one encoding a CYP2D6 mutant having alanine instead of Phe-120 (F120A) and another encoding a mutant having alanine instead of Glu-222 (E222A), were prepared by site-directed mutagenesis and transformed into yeast cells via pGYRI vectors. The enantiomeric BF 1''-hydroxylase activities of the mutants were compared with those of the wild type. When enantiomeric BF 1''-hydroxylase activities at a substrate concentration of 100 microM were compared, the CYP2D6 wild type showed substrate enantioselectivity of (R-BF >> S-BF) and the F120A mutant exhibited substrate enantioselectivity of (R-BF < or = S-BF), whereas the product diastereoselectivity of (1''R-OH-BF << 1''-S-OH-BF) was similar between the wild type and the mutant. The activities of the other mutant (E222A) were much lower than those of the wild type and the F120A mutant, while its substrate enantioselectivity and product diastereoselectivity were the same as those of the wild type. The kinetics demonstrated that apparent K(m) values were similar among the recombinant enzymes, and V(max) values clearly reflected the selectivity described above. These results indicate that Phe-120 has a key role in the enantioselective BF 1''-hydroxylation by CYP2D6.