Importance of Amino Acid Residue 474 for Substrate Specificity of Canine and Human Cytochrome P450 3A Enzymes

Abstract

Canine cytochromes P450 3A12 and 3A26 are identical in sequence at 481 of 503 amino-acid positions but exhibit different substrate specificities. A recent study utilizing chimeric enzymes and site-directed mutagenesis identified three residues (187, 368, and 369) that contribute to differences in steroid hydroxylation and also indicated the presence of additional determinants of specificity among the 44 carboxyl terminal residues. Therefore, three 3A26 multiple mutants (I187T-S368P-V369I-S467P, I187T-S368P-V369I-S474P, and I187T-S368P-V369I-R476K-I477L-T479A-R480Q) were constructed. Insertion of 3A12 residue Pro-474 into 3A26 I187T-S368P-V369I resulted in metabolite profiles with testosterone, androstenedione, and progesterone very similar to 3A12. Substitution of Pro-474 with Ser in P450 3A12 or human 3A4 significantly increased 2β-hydroxylase activity with all three steroids. Residue 474 was also found to be an important contributor to diazepam metabolism by the canine and human enzymes. The results provide further evidence for the role of steric constraints exerted by the enzyme in P450 3A-mediated oxidations.