Functional characterization of eight human cytochrome P450 1A2 gene variants by recombinant protein expression.
Inter-individual variability in cytochrome P450 (CYP)-mediated xenobiotic metabolism is extensive. CYP1A2 is involved in the metabolism of drugs and in the bioactivation of carcinogens. The objective of this study was to functionally characterize eight polymorphic forms of human CYP1A2, namely T83M, S212C, S298R, G299S, I314V, I386F, C406Y and R456H. cDNAs of these variants were constructed and coexpressed in Escherichia coli with human NADPH cytochrome P450 oxidoreductase (CYPOR). All variants showed similar levels of apoprotein and holoprotein expression, except for I386F and R456H, which showed only apoprotein, and both were functionally inactive. The activity of CYP1A2 variants was investigated using 8 substrates, measuring 16 different activity parameters. The resulting heterogeneous activity data set was analyzed together with CYP1A2 wild-type (WT) form, applying multivariate analysis. This analysis indicated that variant G299S is substantially altered in catalytic properties in comparison with WT, whereas variant T83M is slightly but significantly different from the WT. Among CYP1A2 variants, out of the heterogeneous set of eight substrates, carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was the most discriminative compound. In addition, R456 could be identified as an important residue for proper heme binding and stabilization.
Palma
B Brito
BB
Department of Genetics, Faculty of Medical Sciences, Universidade Nova de Lisbon, Lisbon, Portugal.
Silva E Sousa
M
M
Vosmeer
C R
CR
Lastdrager
J
J
Rueff
J
J
Vermeulen
N P E
NP
Kranendonk
M
M
D003001:Cloning, Molecular; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D006418:Heme / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D009602:Nitrosamines / Q000378:metabolism; D011110:Polymorphism, Genetic; D011994:Recombinant Proteins / Q000378:metabolism
D009602:Nitrosamines; D011994:Recombinant Proteins; D006418:Heme; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; D019388:Cytochrome P-450 CYP1A2; D009251:NADPH-Ferrihemoprotein Reductase