Membrane reconstitution of recombinant guinea pig cytochrome P45017alpha and the effects of site-directed mutagenesis on androgen formation.
Although cytochrome P45017alpha 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 P45017alpha produce dehydroepiandrosterone from pregnenolone. Cytochrome P45017alphas 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 P45017alpha with those found in the other animals. The recombinant P45017alphas were expressed in E. coli DH5alpha, purified by column chromatography and incorporated into liposome membranes. The (His)(4) tag in the recombinant P45017alphas had little effect on the interaction with NADPH-P450 reductase in the membranes. The recombinant P45017alphas 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 17alpha-hydroxypregnenolone, androstenedione and dehydroepiandrosterone. The activity for 17alpha-hydroxyprogeterone production was increased significantly by the F344I mutation. The addition of cytochrome b5 did not have much of an effect on the 17alpha-hydroxylation but had a significant effect on androgen production in both the nonmutated and mutated P45017alphas.
Owaki
Akiko
A
Faculty of Integrated Arts and Sciences, Hiroshima University, Higashihiroshima, Japan.
Takamasa
Ayami
A
Yamazaki
Takeshi
T
Kominami
Shiro
S
D000595:Amino Acid Sequence; D000728:Androgens / Q000378:metabolism*; D000735:Androstenedione / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D015786:Cytochromes b5 / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D003687:Dehydroepiandrosterone / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006168:Guinea Pigs; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010088:Oxidoreductases / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism*; D013053:Spectrophotometry; D013254:Steroid 17-alpha-Hydroxylase / Q000737:chemistry / Q000378:metabolism*
D000728:Androgens; D018076:DNA, Complementary; D010455:Peptides; D011994:Recombinant Proteins; D000735:Androstenedione; D003687:Dehydroepiandrosterone; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D013254:Steroid 17-alpha-Hydroxylase; C022433:pregnenolone 17-alpha-hydroxylase