Biochemical analysis of mutations in P450 oxidoreductase.
All microsomal P450s require POR (cytochrome P450 reductase) for catalytic activity. Most of the clinically used drugs are metabolized by a small number of P450s and polymorphisms in the cytochrome P450s are known to cause changes in drug metabolism. We have recently found a number of POR missense mutations in the patients with disordered steroidogenesis. Our initial report described five missense mutations (A284P, R454H, V489E, C566Y and V605F) identified in four patients. We built bacterial expression vectors for each POR variant, purified the membranes expressing normal or variant POR and characterized their activities with cytochrome c and P450c17 assays. We have recently completed an extensive study of the range of POR mutations and characterized the mutants/polymorphisms A112V, T139A, M260V, Y456H, A500V, G536R, L562P, R613X, V628I and F643del from sequencing of patient DNA. We also studied POR variants Y179D, P225L, R313W, G410S and G501R that were available in databases or the published literature. We analysed the mutations with a three-dimensional model of human POR that was based on an essentially similar rat POR with known crystal structure. The missense mutations found in patients with disordered steroidogenesis mapped to functionally important domains of POR and the apparent polymorphisms mapped to less crucial regions. Since a variation in POR can alter the activity of all microsomal P450s, it can also affect the drug metabolism even with a normal P450. Understanding the genetic and biochemical basis of POR-mediated drug metabolism will provide valuable information about possible differences in P450-mediated reactions among the individuals carrying a variant or polymorphic form of POR.
Pandey
A V
AV
Theodor Kocher Institute, University of Bern, Freiestrasse 1, CH-3010 Bern, Switzerland. amit@pandeylab.org
D019943:Amino Acid Substitution; D001665:Binding Sites; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D014644:Genetic Variation; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011110:Polymorphism, Genetic; D011487:Protein Conformation
D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D009251:NADPH-Ferrihemoprotein Reductase