Abstract

Canine cytochromes P-450 3A12 and 3A26 differ by 22 out of 503 amino acid residues. Chimeric constructs and site-directed mutants were used to identify the residues responsible for the much higher rates of steroid hydroxylation by 3A12. Six initial 3A12/3A26 hybrids were generated using convenient restriction sites, and site-directed mutagenesis was used to restore full 3A12 activity to two of the hybrids. One pair of 3A12/3A26 chimeras indicated that the first four residue differences between 3A12 and 3A26 were at least partially responsible for the differences in progesterone hydroxylation. Conversion in one of the hybrids of the Ile-187 residue found in 3A26 to the Thr in 3A12 conferred 3A12 levels of progesterone 6β-hydroxylase activity. Analysis of another chimera identified key residues within an internal PstI fragment (codons 331–459) containing six amino acid residue differences. Subsequent site-directed mutagenesis of 3A26 residues Ser-368 and Val-369 to Pro and Ile, respectively, restored the rate of formation of 6β-hydroxyprogesterone by the hybrid to that of 3A12. The simultaneous conversion of 3A26 residues 187, 368, and 369 to those of 3A12 conferred greater than a third of the progesterone 6β-hydroxylase activity and all of the testosterone and androstenedione 6β-hydroxylase activity of 3A12. Addition of the carboxyl terminal 44 3A12 residues to the 3A26 triple mutant doubled progesterone 6β-hydroxylase activity. This is the first study to use catalytically distinct cytochromes P-450 3A from the same species in the elucidation of structure-function relationships.