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IN VITRO STUDIES ON GENETIC POLYMORPHISM OF CYTOCHROME P450 2A6 (CYP2A6) AND THE INTERACTION OF THE WILD TYPE ISOFORM WITH FLAVONOIDS

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dc.contributor.authorTIONG KAI HUNG
dc.date.accessioned2023-10-06T15:25:47Z
dc.date.available2023-10-06T15:25:47Z
dc.date.issued2009-07
dc.description.abstractThe 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.en_US
dc.identifier.urihttps://hdl.handle.net/20.500.14377/32145
dc.language.isoenen_US
dc.publisherInternational Medical Universityen_US
dc.subjectIn Vitroen_US
dc.subjectPolymorphism, Geneticen_US
dc.subjectCytochrome P-450 Enzyme Systemen_US
dc.subjectMutant Proteinsen_US
dc.subjectAllelesen_US
dc.titleIN VITRO STUDIES ON GENETIC POLYMORPHISM OF CYTOCHROME P450 2A6 (CYP2A6) AND THE INTERACTION OF THE WILD TYPE ISOFORM WITH FLAVONOIDSen_US
dc.typeThesis
dspace.entity.typePublication
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