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Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylation

Naturevolume 399pages670673 (1999) | Download Citation



Enzyme-based chemical transformations typically proceed with high selectivity under mild conditions, and are becoming increasingly important in the pharmaceutical and chemical industries. Cytochrome P450 monooxygenases (P450s) constitute a large family1 of enzymes of particular interest in this regard. Their biological functions, such as detoxification of xenobiotics and steroidogenesis2,3,4,5, are based on the ability to catalyse the insertion of oxygen into a wide variety of compounds6. Such a catalytic transformation might find technological applications in areas ranging from gene therapy and environmental remediation to the selective synthesis of pharmaceuticals and chemicals7,8,9,10. But relatively low turnover rates (particularly towards non-natural substrates), low stability and the need for electron-donating cofactors prohibit the practical use of P450s as isolated enzymes. Here we report the directed evolution11 of the P450 from Pseudomonas putida to create mutants that hydroxylate naphthalene in the absence of cofactors through the ‘peroxide shunt’ pathway12,13 with more than 20-fold higher activity than the native enzyme. We are able to screen efficiently for improved mutants by coexpressing them with horseradish peroxidase, which converts the products of the P450 reaction into fluorescent compounds amenable to digital imaging screening. This system should allow us to select and develop mono- and di-oxygenases into practically useful biocatalysts for the hydroxylation of a wide range of aromatic compounds.

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We thank J. H. Richards for encouragement and support, P. R. Ortiz de Montellano for discussions, and G. Bandara for assistance with purification. This work was supported by the Biotechnology Research and Development Corporation and by the Office of Naval Research. H.J. received partial support from the Korea Research Foundation.

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  1. Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, 91125, California, USA

    • Hyun Joo
    • , Zhanglin Lin
    •  & Frances H. Arnold


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Correspondence to Frances H. Arnold.

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