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Cytochrome P450 BM-3 Evolved by Random and Saturation Mutagenesis as an Effective Indole-Hydroxylating Catalyst

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Abstract

Cytochrome P450 BM-3 with the mutations A74G, F87V, and L188Q could catalyze indole to produce indigo and indirubin. To further enhance this capability, site-directed and random mutageneses on the monooxygenase domain of P450 BM-3 mutant (A74G/F87V/L188Q; 3X) were performed. The mutant libraries created by error-prone polymerase chain reaction were screened using a colorimetric colony-based method on agar plates followed by a spectroscopic assay involving in absorption of indigo at 670 nm and NADPH at 340 nm in microtiter plate. Three mutants (K434R/3X, E435D/3X, and D168N/A225V/K440N/3X) exhibited higher hydroxylation activity toward indole in comparison to parent enzyme. Moreover, using saturation site-directed mutagenesis at amino acid positions 168, 225, 434, 435, and 440, two P450 BM-3 variants (D168H/3X, E435T/3X) with an up to sixfold increase in catalytic efficiency (k cat/K m) were identified, and the mutant D168H/3X acquired higher regioselectivity resulting in more indigo (dimerized 3-hydroxy-indole) compared to parent mutant (93 vs72%).

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Acknowledgements

This work was supported by the joint project of CSC (China)-DAAD (Germany) (2004c33036) and the National Natural Sciences Foundation of China (30570411).

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Authors and Affiliations

  1. Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou, 310027, China

    Hong-mei Li & Le-he Mei

  2. Institute of Food Science and Biological Technology, University of Shanghai for Science and Technology, Shanghai, 200093, China

    Hong-mei Li

  3. Institute of Technical Biochemistry, University of Stuttgart, 70569, Stuttgart, Germany

    V. B. Urlacher & R. D. Schmid

Authors
  1. Hong-mei Li
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  2. Le-he Mei
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  3. V. B. Urlacher
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  4. R. D. Schmid
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Correspondence to Hong-mei Li.

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Li, Hm., Mei, Lh., Urlacher, V.B. et al. Cytochrome P450 BM-3 Evolved by Random and Saturation Mutagenesis as an Effective Indole-Hydroxylating Catalyst. Appl Biochem Biotechnol 144, 27–36 (2008). https://doi.org/10.1007/s12010-007-8002-5

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  • DOI: https://doi.org/10.1007/s12010-007-8002-5

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