title: Role of the conserved threonine 309 in mechanism of oxidation by cytochrome P450 2D6.
abstract: Based on sequence alignments and homology modeling, threonine 309 in cytochrome P450 2D6 (CYP2D6) is proposed to be the conserved I-helix threonine, which is supposed to be involved in dioxygen activation by CYPs. The T309V mutant of CYP2D6 displayed a strong shift from O-dealkylation to N-dealkylation reactions in oxidation of dextromethorphan and 3,4-methylenedioxymethylamphetamine. This may be explained by an elevated ratio of hydroperoxo-iron to oxenoid-iron of the oxygenating species. In consistence, using cumene hydroperoxide, which directly forms the oxenoid-iron, the T309V mutant again selectively catalyzed the O-dealkylation reactions. The changed ratio of oxygenating species can also explain the decreased activity and changed regioselectivity that were observed in 7-methoxy-4-(aminomethyl)-coumarin and bufuralol oxidation, respectively, by the T309V mutant. Interestingly, the T309V mutant always showed a significantly increased, up to 75-fold, higher activity compared to that of the wild-type when using cumene hydroperoxide. These results indicate that T309 in CYP2D6 is involved in maintaining the balance of multiple oxygenating species and thus influences substrate and regioselectivity.
authors.affiliation: Leiden Amsterdam Center for Drug Research (LACDR), Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Peter H J
authors.initials: PH
authors.lastname: Keizers
authors.forename: Loek H M
authors.initials: LH
authors.lastname: Schraven
authors.forename: Chris
authors.initials: C
authors.lastname: de Graaf
authors.forename: Mats
authors.initials: M
authors.lastname: Hidestrand
authors.forename: Magnus
authors.initials: M
authors.lastname: Ingelman-Sundberg
authors.forename: Ben R
authors.initials: BR
authors.lastname: van Dijk
authors.forename: Nico P E
authors.initials: NP
authors.lastname: Vermeulen
authors.forename: Jan N M
authors.initials: JN
authors.lastname: Commandeur
mesh_terms: D019943:Amino Acid Substitution; D019389:Cytochrome P-450 CYP2D6 / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D013912:Threonine; D019389:Cytochrome P-450 CYP2D6