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Preparation of human metabolites of propranolol using laboratory-evolved bacterial cytochromes P450

Authors

  • Christopher R. Otey,

    1. Biochemistry and Molecular Biophysics, California Institute of Technology, Mail code 210-41, Pasadena, California 91125
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  • Geethani Bandara,

    1. Division of Chemistry and Chemical Engineering, California Institute of Technology, Mail code 210-41, Pasadena, California 91125; telephone: (626) 395 4162; fax: (626) 568-8743
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  • James Lalonde,

    1. Division of Chemistry and Chemical Engineering, California Institute of Technology, Mail code 210-41, Pasadena, California 91125; telephone: (626) 395 4162; fax: (626) 568-8743
    Current affiliation:
    1. Codexis, Inc., Biocatalysis and Chemical Development, 200 Penobscot Ave., Redwood City, CA 94063.
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  • Katsuyuki Takahashi,

    1. Division of Chemistry and Chemical Engineering, California Institute of Technology, Mail code 210-41, Pasadena, California 91125; telephone: (626) 395 4162; fax: (626) 568-8743
    Current affiliation:
    1. Mitsui Chemicals, Inc., 1144 Togo, Mobara-shi, Chiba 297-0017, Japan.
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  • Frances H. Arnold

    Corresponding author
    1. Biochemistry and Molecular Biophysics, California Institute of Technology, Mail code 210-41, Pasadena, California 91125
    2. Division of Chemistry and Chemical Engineering, California Institute of Technology, Mail code 210-41, Pasadena, California 91125; telephone: (626) 395 4162; fax: (626) 568-8743
    • Biochemistry and Molecular Biophysics, California Institute of Technology, Mail code 210-41, Pasadena, California 91125
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  • Geethani Bandara and Christopher R. Otey contributed equally to this study.

Abstract

Testing the toxicities and biological activities of the human metabolites of drugs is important for development of safe and effective pharmaceuticals. Producing these metabolites using human cytochrome P450s is difficult, however, because the human enzymes are costly, poorly stable, and slow. We have used directed evolution to generate variants of P450 BM3 from Bacillus megaterium that function via the “peroxide shunt” pathway, using hydrogen peroxide in place of the reductase domain, oxygen and NADPH. Here, we report further evolution of the P450 BM3 heme domain peroxygenase to enhance production of the authentic human metabolites of propranolol by this biocatalytic route. This system offers a versatile, cost-effective, and scaleable route to the synthesis of drug metabolites. © 2005 Wiley Periodicals, Inc.

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