title: Directed-evolution analysis of human cytochrome P450 2A6 for enhanced enzymatic catalysis.
abstract: Cytochrome P450 2A6 (P450 2A6) is the major enzyme responsible for the oxidation of coumarin, nicotine, and tobacco-specific nitrosamines in human liver. In this study, the catalytic turnover of coumarin oxidation was improved by directed-evolution analysis of P450 2A6 enzyme. A random mutant library was constructed using error-prone polymerase chain reaction (PCR) of the open reading frame of the P450 2A6 gene and individual mutant clones were screened for improved catalytic activity in analysis of fluorescent coumarin 7-hydroxylation. Four consecutive rounds of random mutagenesis and screening were performed and catalytically enhanced mutants were selected in each round of screening. The selected mutants showed the sequentially accumulated mutations of amino acid residues of P450 2A6: B1 (F209S), C1 (F209S, S369G), D1 (F209S, S369G, E277K), and E1 (F209S, S369G, E277K, A10V). E1 mutants displayed approximately 13-fold increased activity based on fluorescent coumarin hydroxylation assays at bacterial whole cell level. Steady-state kinetic parameters for coumarin 7-hydroxylation and nicotine oxidation were measured in purified mutant enzymes and indicated catalytic turnover numbers (kcat) of selected mutants were enhanced up to sevenfold greater than wild-type P450 2A6. However, all mutants displayed elevated Km values and therefore catalytic efficiencies (kcat/Km) were not improved. The increase in Km values was partially attributed to reduction in substrate binding affinities measured in the analysis of substrate binding titration. The structural analysis of P450 2A6 indicates that F209S mutation is sufficient to affect direct interaction of substrate at the active site.
authors.affiliation: a Department of Biological Sciences , Konkuk University , Seoul , Korea.
authors.forename: Hwayoun
authors.initials: H
authors.lastname: Lee
authors.forename: Joo-Hwan
authors.initials: JH
authors.lastname: Kim
authors.forename: Songhee
authors.initials: S
authors.lastname: Han
authors.forename: Young-Ran
authors.initials: YR
authors.lastname: Lim
authors.forename: Hyoung-Goo
authors.initials: HG
authors.lastname: Park
authors.forename: Young-Jin
authors.initials: YJ
authors.lastname: Chun
authors.forename: Sung-Woo
authors.initials: SW
authors.lastname: Park
authors.forename: Donghak
authors.initials: D
authors.lastname: Kim
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D003374:Coumarins / Q000378:metabolism; D065637:Cytochrome P-450 CYP2A6 / Q000235:genetics / Q000378:metabolism*; D019020:Directed Molecular Evolution; D006801:Humans; D006900:Hydroxylation; D007093:Imidazoles / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D016296:Mutagenesis; D009154:Mutation; D009538:Nicotine / Q000378:metabolism; D009602:Nitrosamines / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D003374:Coumarins; D007093:Imidazoles; D009602:Nitrosamines; D011994:Recombinant Proteins; C517283:coumarin 7; D009538:Nicotine; D065637:Cytochrome P-450 CYP2A6