NDLI: Alteration of the substrate specificity of cytochrome P450 CYP199A2 by site-directed mutagenesis.

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Alteration of the substrate specificity of cytochrome P450 CYP199A2 by site-directed mutagenesis.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Furuya, Toshiki Shitashima, Yoh Kino, Kuniki
Description	Country affiliation: Japan Author Affiliation: Furuya T ( Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan.); Shitashima Y ( Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan.); Kino K ( Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan. Electronic address: kkino@waseda.jp.)
Abstract	CYP199A2, a member of the cytochrome P450 family, is a monooxygenase that specializes in the oxidation of aromatic carboxylic acids. The crystal structure of CYP199A2 determined by Bell et al. (J. Mol. Biol., 383, 561-574, 2008) suggested that the S97 and S247 residues conferred the substrate specificity on this enzyme through interaction between the hydroxy side chains of these Ser residues and the carboxy group of the substrates. In this study, we attempted to design and construct CYP199A2 mutants that recognize hydroxy aromatic compounds as substrates by protein engineering. We speculated that substitution of the S97 and S247 residues with acidic amino acids Asp and Glu, which have carboxy side chains, would provide CYP199A2 mutants that recognize hydroxy aromatic compounds instead of aromatic carboxylic acids. The S97 and S247 residues were substituted with Asp and Glu using site-directed mutagenesis. In whole-cell assays with p-methylbenzylalcohol and phenol as hydroxy aromatic substrates, the S247D mutant regioselectively oxidized these compounds to 1,4-benzenedimethanol and hydroquinone, respectively, although the wild-type enzyme exhibited no oxidation activity for these compounds. Furthermore, the S97D, S247D, and S247E mutants acquired oxidation activity for p-cresol. Especially, the S247D mutant rapidly oxidized p-cresol; the whole cells expressing the S247D mutant completely converted 1 mM p-cresol to p-hydroxybenzylalcohol in only 30 min. These results also clearly demonstrate that S97 and S247 are important residues that control the substrate specificity of CYP199A2.
File Format	HTM / HTML
ISSN	13891723
Issue Number	1
Volume Number	119
e-ISSN	13474421
Journal	Journal of Bioscience and Bioengineering
Language	English
Publisher	Elsevier
Publisher Date	2015-01-01
Publisher Place	Japan
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Discipline Biomedical Engineering Discipline Microbiology Cytochrome P-450 Enzyme System Genetics Metabolism Mutagenesis, Site-directed Carboxylic Acids Cresols Chemistry Escherichia Coli Hydroxylation Models, Molecular Mutation Oxidation-reduction Substrate Specificity Journal Article
Content Type	Text
Resource Type	Article
Subject	Bioengineering Applied Microbiology and Biotechnology Biotechnology

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