Metabolism of 7‐ethoxycoumarin, safrole, flavanone and hydroxyflavanone by cytochrome P450 2A6 variants

Abstract

CYP 2A6 is a human enzyme that metabolizes many xenobiotics including coumarin, indole, nicotine and carcinogenic nitrosamines. The gene for CYP2A6 is polymorphic. There are few data available to clarify the relationship between P450 genetic variants and the metabolism of materials in food. The CYP 2A6 wild-type protein and 13 mutants (CYP2A6.1, CYP2A6.2, CYP2A6.5, CYP2A6.6, CYP2A6.7, CYP2A6.8, CYP2A6.11, CYP2A6.15, CYP2A6.16, CYP2A6.17, CYP2A6.18, CYP2A6.21, CYP2A6.23 and CYP2A6.25) were co-expressed with NADPH-cytochrome P450 reductase in E. coli. The hydroxylase activities toward 7-ethoxycoumarin, coumarin, safrole, flavanone and hydroxyflavanone were examined. Ten types of CYP2A6 variants except for CYP2A6.2, CYP2A6.5 and CYP2A6.6 showed Soret peaks (450 nm) typical of P450 in the reduced CO-difference spectra and had 7-ethoxycoumarin O-deethylase activities. CYP2A6.15 and CYP2A6.18 showed higher activities for safrole 1'-hydroxylation than CYP2A6.1. CYP2A6.25 and CYP2A6.7 had lower safrole 1'-hydroxylase activities. CYP2A6.7 had lower flavanone 6- and 2'-hydroxylase activities, whereas CYP2A6.25 had higher 6-hydroxylase activity and lower 2'-hydroxylase activity. Hydroxyflavanone was metabolized by CYP2A6.25, but was not metabolized by wild-type CYP2A6.1. These results indicate that CYP2A6.25 possessed new substrate specificity toward flavonoids.

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

“…CYP2A6*7 contains a missense mutation, 6558G>A, which produces an Ile471Thr amino acid change that decreases the enzymatic activity to metabolize some substrates for in vivo and in vitro assays (Ariyoshi et al, 2001[7]; Uno et al, 2013[138]; Xu et al, 2002[147]).…”

“…CYP2A6*25 is the result of several point mutations, some of them in 5'UTR: -1301A>C, -1289G>A and -745A>G, also 22C>T, 51G, 768A>T, 1620T>C, 1672T>C, 2296C>T, 2483G>A, 2605G>A, 2921G>A, 2994T>C, 4636A>C, 5668A, 6586T>C, 6692C>G and 7160A>G that result in the amino acid change of Phe118Leu (Mwenifumbo et al, 2008[87]), it has been shown that its enzymatic activity is decreased in some substrates (Ho et al, 2009[49]; Mwenifumbo et al, 2008[87]; Uno et al, 2013[138]).…”

Point mutation of cytochrome P450 2A6 (a polymorphic variant CYP2A6.25) confers new substrate specificity towards flavonoids

“…In addition, two expression vectors, CYP2A6.1 and CYP2A6.25 were obtained as described previously [20]. In addition, two expression vectors, CYP2A6.1 and CYP2A6.25 were obtained as described previously [20].…”

“…Many CYP2A6 genetic variants, which showed reduced enzymatic activity, are associated with slower metabolism of nicotine and cotinine [12][13][14][15]. In a previous study, it was found that CYP2A6.25, which comprises an amino acid substitution of F118L, shows a decrease in coumarin 7hydroxylation and safrole hydroxylation [20]. There are few reports clarifying the contributions of the specific amino acid residues that are mutated in these CYP2A6 variants.…”

“…In addition, flavanone is the precursor to all flavonoid structures and has important antioxidant activities [25][26][27][28]. In a previous study, it was found that CYP2A6.25, which comprises an amino acid substitution of F118L, shows higher flavanone 6-hydroxylase activity and lower 2′-hydroxylase activity than a wild type CYP2A6.1; and metabolize 4′-hydroxyflavanone, which is not metabolized by CYP2A6.1 [20]. As yet, there are no reports clarifying the CYP amino acid residues essential for the metabolism of flavonoids, nor the relationship between CYP polymorphisms and their effect on flavonoid hydroxylation.…”