Abstract:
The work described in this study aimed to clone and express CYP2A6 wild type
and mutant proteins in bacterial system, to use the expressed proteins to investigate the
structural and functional consequences of reported allele CYP2A6*15 (Lys194Glu),
CYP2A6*16 (Arg203Ser), CYP2A6*21 (Lys476Arg) and CYP2A6*22 (Asp158Glu
and Leu160Ile) and to characterize potency and mechanism of modulation of naturally
occurring flavonoids on wild type CYP2A6 activity. Human wild type CYP2A6 was
initially isolated from the human liver total RNA and four mutant alleles were
successfully generated. Wild type and mutant CYP2A6 proteins were expressed at
comparable level as revealed by Western blotting analyses. Interestingly, except for
CYP2A6*22, all mutant CYP2A6 proteins exhibited catalytically active enzyme
activities as evidenced from the high Soret peak at 450 nm in the absorbance spectral
analyses and comparable activities from fluorescence-based coumarin 7-hydroxylase
assay. Such observations indicated that amino acid mutations in the primary sequence
of the respective CYP2A6 alleles did not affect the structural stability and functional
catalytic activity in coumarin 7-hydroxylation. CYP2A6*22 exhibited only half the
activity of the wild-type indicating that Asp158Glu and Leu160Ile substitutions had
caused compromised activity in this allele. Screening of flavonoid compounds on
coumarin 7-hydroxylase assay indicated different potency of inhibition on the wild type
CYP2A6 activity. Among the twenty-three compounds tested, members were from
flavonol group of hydroxylated flavonoids were the most inhibitory, with myricetin
being the most potent inhibitor followed by quercetin, galangin and kaempferol. Further
exploration of the inhibition mechanism of these compounds revealed that myricetin,
galangin and kaempferol exhibited mixed-type of inhibition pattern while quercetin was
observed to portrait competitive mode of inhibition in coumarin 7-hydroxylation.
Structure-function analyses revealed that degree of inhibition was closely related to the
number and location of hydroxyl groups, glycosylation of these free hydroxyl groups,
degree of saturation of the flavane nucleus as well as the presence of the alkoxylated function.
