Substrate-specific modulation of CYP3A4 activity by genetic variants of cytochrome P450 oxidoreductase.
OBJECTIVE
CYP3A4 receives electrons from P450 oxidoreductase (POR) to metabolize about 50% of clinically used drugs. There is substantial inter-individual variation in CYP3A4 catalytic activity that is not explained by CYP3A4 genetic variants. CYP3A4 is flexible and distensible, permitting it to accommodate substrates varying in shape and size. To elucidate the mechanisms of variability in CYP3A4 catalysis, we examined the effects of genetic variants of POR, and explored the possibility that substrate-induced conformational changes in CYP3A4 differentially affect the ability of POR variants to support catalysis.


METHODS
We expressed human CYP3A4 and four POR variants (Q153R, A287P, R457H, A503 V) in bacteria, reconstituted them in vitro and measured the Michaelis constant and maximum velocity with testosterone, midazolam, quinidine and erythromycin as substrates.


RESULTS
POR A287P and R457H had low activity with all substrates; Q153R had 76-94% of wild-type (WT) activity with midazolam and erythromycin, but 129-150% activity with testosterone and quinidine. The A503 V polymorphism reduced the CYP3A4 activity to 61-77% of WT with testosterone and midazolam, but had nearly WT activity with quinidine and erythromycin.


CONCLUSIONS
POR variants affect CYP3A4 activities. The impact of a POR variant on catalysis by CYP3A4 is substrate-specific, probably because of substrate-induced conformational changes in CYP3A4.
Agrawal
Vishal
V
Department of Pediatrics, University of California, San Francisco, California 94143-0978, USA.
Choi
Ji Ha
JH
Giacomini
Kathleen M
KM
Miller
Walter L
WL
D055162:Biocatalysis; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D004917:Erythromycin / Q000737:chemistry / Q000378:metabolism; D014644:Genetic Variation; D006801:Humans; D007700:Kinetics; D008874:Midazolam / Q000737:chemistry / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011802:Quinidine / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013739:Testosterone / Q000737:chemistry / Q000378:metabolism
D050505:Mutant Proteins; D013739:Testosterone; D004917:Erythromycin; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D009251:NADPH-Ferrihemoprotein Reductase; D011802:Quinidine; D008874:Midazolam