Membrane reconstitution of recombinant guinea pig cytochrome P45017α and the effects of site-directed mutagenesis on androgen formation

Abstract

Although cytochrome P45017α catalyzes the formation of androgen from both pregnenolone and progesterone, the production of androstenedione from progesterone is a major pathway in the guinea pig, rat, mouse, and hamster. In contrast, human, bovine and sheep P45017α produce dehydroepiandrosterone from pregnenolone. Cytochrome P45017αs from all of these animals have high homology in the amino acid sequence around the 340–370 region. To investigate the substrate preferences for androgen production, we replaced a few amino acids in the 340–370 region of guinea pig P45017α with those found in the other animals. The recombinant P45017αs were expressed in E. coli DH5α, purified by column chromatography and incorporated into liposome membranes. The (His) 4 tag in the recombinant P45017αs had little effect on the interaction with NADPH–P450 reductase in the membranes. The recombinant P45017αs with a single-position mutation of F344I, H349R or M352L and with double-position mutations of F344I and H349R and triple-position mutations showed decreases in the production of 17α-hydroxypregnenolone, androstenedione and dehydroepiandrosterone. The activity for 17α-hydroxyprogeterone production was increased significantly by the F344I mutation. The addition of cytochrome b5 did not have much of an effect on the 17α-hydroxylation but had a significant effect on androgen production in both the nonmutated and mutated P45017αs.