title: Metabolism of bilirubin by human cytochrome P450 2A6.
abstract: The mouse cytochrome P450 (CYP) 2A5 has recently been shown to function as hepatic "Bilirubin Oxidase" (Abu-Bakar, A., et al., 2011. Toxicol. Appl. Pharmacol. 257, 14-22). To date, no information is available on human CYP isoforms involvement in bilirubin metabolism. In this paper we provide novel evidence for human CYP2A6 metabolising the tetrapyrrole bilirubin. Incubation of bilirubin with recombinant yeast microsomes expressing the CYP2A6 showed that bilirubin inhibited CYP2A6-dependent coumarin 7-hydroxylase activity to almost 100% with an estimated K(i) of 2.23 μM. Metabolite screening by a high-performance liquid chromatography/electrospray ionisation mass spectrometry indicated that CYP2A6 oxidised bilirubin to biliverdin and to three other smaller products with m/z values of 301, 315 and 333. Molecular docking analyses indicated that bilirubin and its positively charged intermediate interacted with key amino acid residues at the enzyme's active site. They were stabilised at the site in a conformation favouring biliverdin formation. By contrast, the end product, biliverdin was less fitting to the active site with the critical central methylene bridge distanced from the CYP2A6 haem iron facilitating its release. Furthermore, bilirubin treatment of HepG2 cells increased the CYP2A6 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A6 compared to cells treated only with CHX. Collectively, the observations indicate that the CYP2A6 may function as human "Bilirubin Oxidase" where bilirubin is potentially a substrate and a regulator of the enzyme.
authors.affiliation: The University of Queensland, National Research Centre for Environmental Toxicology-Entox, 4072 Brisbane, Queensland, Australia. a.abubakar@uq.edu.au
authors.forename: A'edah
authors.initials: A
authors.lastname: Abu-Bakar
authors.forename: Dionne M
authors.initials: DM
authors.lastname: Arthur
authors.forename: Anna S
authors.initials: AS
authors.lastname: Wikman
authors.forename: Minna
authors.initials: M
authors.lastname: Rahnasto
authors.forename: Risto O
authors.initials: RO
authors.lastname: Juvonen
authors.forename: Jouko
authors.initials: J
authors.lastname: Vepsäläinen
authors.forename: Hannu
authors.initials: H
authors.lastname: Raunio
authors.forename: Jack C
authors.initials: JC
authors.lastname: Ng
authors.forename: Matti A
authors.initials: MA
authors.lastname: Lang
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D001663:Bilirubin / Q000378:metabolism*; D001664:Biliverdine / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003513:Cycloheximide / Q000494:pharmacology; D065637:Cytochrome P-450 CYP2A6; D006207:Half-Life; D056945:Hep G2 Cells; D006801:Humans; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism*; D008958:Models, Molecular; D012333:RNA, Messenger / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D012333:RNA, Messenger; D003513:Cycloheximide; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; D065637:Cytochrome P-450 CYP2A6; D001664:Biliverdine; D001663:Bilirubin