A single amino acid mutation (Ser180----Cys) determines the polymorphism in cytochrome P450g (P4502C13) by altering protein stability.
Cytochrome P450g is polymorphic in the male rat. This polymorphism is characterized by a 20-40-fold difference in the hepatic content of P450g in the two phenotypes. Sequencing of cDNAs from high (+g) and low (-g) phenotype rats has shown that the low phenotype is due to a defective mRNA containing nine base mutations encoding 7 amino acid substitutions. To determine the role of these structural changes in the phenotypic expression of P450g, we altered each of these residues by site-directed mutagenesis in the present studies and expressed the normal and mutant cDNAs in Saccharomyces cerevisiae. P450+g protein was expressed at a level 4-6-fold higher than that of P450-g in yeast cells, despite the presence of identical mRNA levels. This difference in protein expression approaches the difference seen in the rat. A single amino acid change from Ser180 in P450+g to Cys in P450-g, in a highly conserved region in the P4502C subfamily, was found to be solely responsible for the phenotypic differences in expression of P450g. Protein half-life studies demonstrated that this mutation increases the degradation of P450g. This is the first example of a single amino acid substitution which alters the phenotypic expression of a P450 protein by affecting its stability.
Faletto
M B
MB
Laboratory of Biochemical Risk Analysis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
Linko
P
P
Goldstein
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003545:Cysteine; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004795:Enzyme Stability; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010641:Phenotype; D010957:Plasmids; D011110:Polymorphism, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012694:Serine
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012694:Serine; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D003545:Cysteine