Functional Characterization of Cytochrome P450 2A6 Allelic VariantsCYP2A6*15,CYP2A6*16,CYP2A6*21, andCYP2A6*22

Abstract

ABSTRACT:Variation in CYP2A6 levels and activity can be attributed to genetic polymorphism and, thus, functional characterization of allelic variants is necessary to define the importance of CYP2A6 polymorphism in humans. The aim of the present study was to investigate the reported alleles CYP2A6*15, CYP2A6*16, CYP2A6*21, and CYP2A6*22, in terms of the functional consequences of their mutations on the enzyme catalytic activity. With use of the wild-type CYP2A6 cDNA as template, site-directed mutagenesis was performed to introduce nucleotide changes encoding K194E substitution in CYP2A6*15, R203S substitution in CYP2A6*16, K476R substitution in CYP2A6*21, and concurrent D158E and L160I substitutions in CYP2A6*22. Upon sequence verification, the CYP2A6 wild-type and mutant constructs were individually coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. A kinetic study using a coumarin 7-hydroxylase assay indicated that CYP2A6*15 exhibited higher V max than the wild type, whereas all mutant constructs, except for variant CYP2A6*16, exhibited higher K m values. Analysis of the V max /K m ratio revealed that all mutants demonstrated 0.85-to 1.05-fold differences from the wild type, with the exception of variant CYP2A6*22, which only portrayed 39% of the wild-type intrinsic clearance. These data suggested that individuals carrying the CYP2A6*22 allele are likely to have lower metabolism of CYP2A6 substrate than individuals expressing CYP2A6*15, CYP2A6*16, CYP2A6*21, and the wild type.

Effect of CYP2A6 Genetic Polymorphism on the Metabolic Conversion of Tegafur to 5-Fluorouracil and Its Enantioselectivity

“…Rabbit anti-CYP2A6 and anti-CPR polyclonal antibodies detected CYP2A6 and CPR expressed in Sf9 cells with molecular masses of approximately 50 and 80 kDa, respectively, which corresponded to known molecular weights of these enzymes (Vermilion and Coon, 1978;Tiong et al, 2010). The molecular weight of the positive control for CYP2A6 (Fig.…”

“…Despite the fact that CYP2A6 is one of major polymorphic isoforms, as described above, the functional impact of its SNPs on the enantioselective metabolism of chiral substrates has not been examined (Fukami et al, 2005;Kimura et al, 2005;Tiong et al, 2010;Han et al, 2012). FT has been approved in several oral formulations for the treatment of various cancers and is thereby one of the most frequently prescribed drugs for cancer chemotherapy in Asia (Watanabe, 2013).…”

Distribution of polymorphic variants of CYP2A6 and their involvement in nicotine addiction

“…However, this enzyme does not show differences in substrate metabolism (Tiong et al, 2014[134]; 2010[135]).…”

“…CYP2A6*16 has a missense mutation at 2161C>A, which makes an amino acid change of Arg203Ser (Kiyotani et al, 2002[64]); however, it does not cause a defect in the enzymatic activity (Ho et al, 2008[50]; Nakajima et al, 2006[91]; Tiong et al, 2014[134], 2010[135]).…”

“…However, the functional effect of the enzyme is still under discussion because it has been reported that in vivo assays show differences according to the study population (Al Koudsi et al, 2006[3]; Mwenifumbo et al, 2008[87]) and in vitro assays show normal enzymatic activity (Tiong et al, 2014[134], 2010[135]).…”