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

Toshiki Furuya, Yoh Shitashima, Kuniki Kino

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    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 etal. (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 1mM p-cresol to p-hydroxybenzylalcohol in only 30min. These results also clearly demonstrate that S97 and S247 are important residues that control the substrate specificity of CYP199A2.

    Original languageEnglish
    Pages (from-to)47-51
    Number of pages5
    JournalJournal of Bioscience and Bioengineering
    Issue number1
    Publication statusPublished - 2015 Jan 1



    • CYP199A2
    • Cytochrome P450
    • Hydroxylation
    • Monooxygenase
    • Site-directed mutagenesis

    ASJC Scopus subject areas

    • Biotechnology
    • Applied Microbiology and Biotechnology
    • Bioengineering

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