Structure-function relationships of rat liver CYP3A9 to its human liver orthologs: site-directed active site mutagenesis to a progesterone dihydroxylase.
CYP3A9 is an estrogen-inducible ortholog of human liver CYP3A4 with 76.5% sequence identity to CYP3A4. Unlike CYP3A4, it is a very poor testosterone 6beta- and 2beta-hydroxylase, but a relatively better catalyst of progesterone monohydroxylation largely at 6beta, 16alpha, and 21 positions with negligible 6beta, 21-dihydroxylation. We reasoned that such differences in substrate catalyses must be due to differences in the active site architecture of each CYP3A enzyme. Indeed, alignment of CYP3A4 substrate recognition sites (SRSs) with the corresponding regions of CYP3A9 sequence revealed that of the 22 fully divergent residues, 4 reside in SRS regions [P107N (SRS-1), M371G (SRS-5), and L479K and G480Q (SRS-6)]. Accordingly, we substituted these and other divergent CYP3A9 SRS residues with the corresponding residues of CYP3A4 and/or CYP3A5. Our findings of the influence of these site-directed mutations of the CYP3A9 active site on its catalysis of testosterone and three other established but structurally different CYP3A substrates (progesterone, imipramine, and carbamazepine) are described. These findings revealed that some mutations (N107P, N107S, V207T, G371M, and Q480G) not only improved the ability of CYP3A9 to hydroxylate testosterone at the 6beta and 2beta positions, but also converted it into a robust progesterone 6beta, 21-dihydroxylase. The latter in the case of CYP3A9N107P was accompanied by a shift from sigmoidal to hyperbolic enzyme-substrate kinetics. In contrast, the catalytic potential of CYP3A9 mutants K206N, K206S, M240V, and K479L/Q480G was either relatively unchanged or negligible to nonexistent. Together these findings attest to the unique substrate-active site fit of each CYP3A enzyme.
Xue
Linlong
L
Department of Cellular and Molecular Pharmacology, University of California, Box 0450, San Francisco 94143-0450, USA.
Zgoda
Victor G
VG
Arison
Byron
B
Correia
Maria Almira
MA
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001665:Binding Sites; D002384:Catalysis; D051544:Cytochrome P-450 CYP3A; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007099:Imipramine / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011522:Protons; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013250:Steroid Hydroxylases / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013997:Time Factors
D008565:Membrane Proteins; D011522:Protons; D011994:Recombinant Proteins; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C465260:Cyp3a2 protein, rat; C415842:Cyp3a9 protein, rat; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D007099:Imipramine