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Open AccessArticle

Directed Evolution of P450 BM3 towards Functionalization of Aromatic O-Heterocycles

1
Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany
2
DWI-Leibniz-Institut für Interaktive Materialien e.V., Forckenbeckstraße 50, 52074 Aachen, Germany
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2019, 20(13), 3353; https://doi.org/10.3390/ijms20133353
Received: 5 June 2019 / Revised: 28 June 2019 / Accepted: 1 July 2019 / Published: 8 July 2019
The O-heterocycles, benzo-1,4-dioxane, phthalan, isochroman, 2,3-dihydrobenzofuran, benzofuran, and dibenzofuran are important building blocks with considerable medical application for the production of pharmaceuticals. Cytochrome P450 monooxygenase (P450) Bacillus megaterium 3 (BM3) wild type (WT) from Bacillus megaterium has low to no conversion of the six O-heterocycles. Screening of in-house libraries for active variants yielded P450 BM3 CM1 (R255P/P329H), which was subjected to directed evolution and site saturation mutagenesis of four positions. The latter led to the identification of position R255, which when introduced in the P450 BM3 WT, outperformed all other variants. The initial oxidation rate of nicotinamide adenine dinucleotide phosphate (NADPH) consumption increased ≈140-fold (WT: 8.3 ± 1.3 min−1; R255L: 1168 ± 163 min−1), total turnover number (TTN) increased ≈21-fold (WT: 40 ± 3; R255L: 860 ± 15), and coupling efficiency, ≈2.9-fold (WT: 8.8 ± 0.1%; R255L: 25.7 ± 1.0%). Computational analysis showed that substitution R255L (distant from the heme-cofactor) does not have the salt bridge formed with D217 in WT, which introduces flexibility into the I-helix and leads to a heme rearrangement allowing for efficient hydroxylation. View Full-Text
Keywords: protein engineering; directed evolution; P450; monooxygenases aromatic heterocycles; hydroxylation; molecular modeling protein engineering; directed evolution; P450; monooxygenases aromatic heterocycles; hydroxylation; molecular modeling
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Santos, G.A.; Dhoke, G.V.; Davari, M.D.; Ruff, A.J.; Schwaneberg, U. Directed Evolution of P450 BM3 towards Functionalization of Aromatic O-Heterocycles. Int. J. Mol. Sci. 2019, 20, 3353.

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