1. Cytochrome P450 BM-3 evolved by random and saturation mutagenesis as an effective indole-hydroxylating catalyst.
- Author
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Li HM, Mei LH, Urlacher VB, and Schmid RD
- Subjects
- Bacillus megaterium enzymology, Bacillus megaterium genetics, Bacterial Proteins chemistry, Base Sequence, Catalytic Domain genetics, Cytochrome P-450 Enzyme System chemistry, DNA, Bacterial genetics, Directed Molecular Evolution, Hydroxylation, Indigo Carmine, Kinetics, Mixed Function Oxygenases chemistry, Models, Molecular, Mutagenesis, Mutagenesis, Site-Directed, NADPH-Ferrihemoprotein Reductase, Protein Conformation, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Bacterial Proteins genetics, Bacterial Proteins metabolism, Cytochrome P-450 Enzyme System genetics, Cytochrome P-450 Enzyme System metabolism, Indoles metabolism, Mixed Function Oxygenases genetics, Mixed Function Oxygenases metabolism
- 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 (kcat/Km) were identified, and the mutant D168H/3X acquired higher regioselectivity resulting in more indigo (dimerized 3hydroxy-indole) compared to parent mutant (93 vs 72%).
- Published
- 2008
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