title: Expansion of substrate specificity of cytochrome P450 2A6 by random and site-directed mutagenesis.
abstract: The natural product indole is a substrate for cytochrome P450 2A6. Mutagenesis of P450 2A6 was done to expand its capability in the oxidization of bulky substituted indole compounds, which are not substrates for the wild-type enzyme or the double mutant L240C/N297Q, as determined in our previous work (Wu, Z.-L., Aryal, P., Lozach, O., Meijer, L., and Guengerich, F. P. (2005) Chem. Biodivers. 2, 51-65). Error-prone PCR and site-directed mutagenesis led to the identification of two critical amino acid residue changes (N297Q and I300V) that achieve the purpose. The new mutant (N297Q/I300V) was able to oxidize both 4- and 5-benzyloxy(OBzl)indoles to form colored products. Both changes were required for oxidation of these bulky substrates. The colored product derived from 5-OBzl-indole was mainly 5,5'-di-OBzl-indirubin, whereas the dominant blue dye isolated upon incubations with 4-OBzl-indole was neither an indigo nor an indirubin. Two-dimensional NMR experiments led to assignment of the structure as 4-OBzl-2-(4'-OBzl-1',7'-dihydro-7'-oxo-6'H-indol-6'-ylidene)indolin-3-one, in which a pyrrole ring and a benzene ring are connected with a double bond instead of the pyrrole-pyrrole connection of other indigoids. Monomeric oxidation products were also isolated and characterized; three phenols (4-OBzl-1H-indol-5-ol, 4-OBzl-1H-indol-6-ol, and 4-OBzl-1H-indol-7-ol) and one quinone (4-OBzl-1H-indole-6,7-dione, the postulated immediate precursor of the final blue dye) were identified. The results are interpreted in the context of a crystal structure of a P450 2A6-coumarin complex. The I300V change opens an additional pocket to accommodate the OBzl bulk. The N2297Q change is postulated to generate a hydrogen bond between Gln and the substrate oxygen. Thus, the substrate specificity of P450 2A6 was expanded, and new products were obtained in this study.
authors.affiliation: Department of Biochemistry and the Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
authors.forename: Zhong-Liu
authors.initials: ZL
authors.lastname: Wu
authors.forename: Larissa M
authors.initials: LM
authors.lastname: Podust
authors.forename: F Peter
authors.initials: FP
authors.lastname: Guengerich
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001593:Benzyl Compounds; D001665:Binding Sites; D003374:Coumarins / Q000378:metabolism; D003460:Crystallization; D065637:Cytochrome P-450 CYP2A6; D015723:Gene Library; D006801:Humans; D006860:Hydrogen Bonding; D007211:Indoles / Q000032:analysis / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D001593:Benzyl Compounds; D003374:Coumarins; D007211:Indoles; D011994:Recombinant Proteins; C062517:4-chloroindole; C030374:indole; C030123:coumarin; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; D065637:Cytochrome P-450 CYP2A6