Cytochrome P450 BM-3 Evolved by Random and Saturation Mutagenesis as an Effective Indole-Hydroxylating Catalyst |
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| Authors: | Hong-mei Li Le-he Mei V. B. Urlacher R. D. Schmid |
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| Affiliation: | (1) Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou, 310027, China;(2) Institute of Food Science and Biological Technology, University of Shanghai for Science and Technology, Shanghai, 200093, China;(3) Institute of Technical Biochemistry, University of Stuttgart, 70569 Stuttgart, Germany |
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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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| Keywords: | Cytochrome P450 monooxygenase Error-prone PCR Indole Indigo Saturation mutagenesis P450 BM-3 |
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