PMID: 18415984Apr 17, 2008Paper

Cytochrome P450 BM-3 evolved by random and saturation mutagenesis as an effective indole-hydroxylating catalyst

Applied Biochemistry and Biotechnology
Hong-mei LiR D Schmid

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%).

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Citations

Aug 15, 2012·Proceedings of the National Academy of Sciences of the United States of America·Hsin H Kuo, A Grant Mauk
Oct 19, 2010·BMC Structural Biology·Demet SirimJürgen Pleiss
Apr 28, 2011·Applied Microbiology and Biotechnology·Patrizia Di GennaroGuido Sello
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