Use of chimeric enzymes and site-directed mutagenesis for identification of three key residues responsible for differences in steroid hydroxylation between canine cytochromes P-450 3A12 and 3A26

Canine cytochromes P-450 3A12 and 3A26 differ by 22 out of 503 amino acid r esidues. Chimeric constructs and site-directed mutants were used to identif y the residues responsible for the much higher rates of steroid hydroxylati on by 3A12. Six initial 3A12/3A26 hybrids were generated using convenient r estriction sites, and site-directed mutagenesis was used to restore full 3A 12 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 leas t partially responsible for the differences in progesterone hydroxylation. Conversion in one of the hybrids of the lie-187 residue found in 3A26 to th e Thr in 3A12 conferred 3A12 levels of progesterone 6 beta-hydroxylase acti vity. Analysis of another chimera identified key residues within an interna l PstI fragment (codons 331-459) containing six amino acid residue differen ces. Subsequent site-directed mutagenesis of 3A26 residues Ser-368 and Val- 369 to Pro and lie, respectively, restored the rate of formation of 6 beta- hydroxyprogesterone by the hybrid to that of 3A12. The simultaneous convers ion of 3A26 residues 187, 368, and 369 to those of 3A12 conferred greater t han a third of the progesterone GP-hydroxylase activity and all of the test osterone and androstenedione 6 beta-hydroxylase activity of 3A12, Addition of the carboxyl terminal 44 3A12 residues to the 3A26 triple mutant doubled progesterone 6 beta-hydroxylase activity. This is the first study to use c atalytically distinct cytochromes P-450 3A from the same species in the elu cidation of structure-function relationships.