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

• Published in: International journal of molecular sciences 2019-07, Vol.20 (13), p.3353
• Main Authors: Santos, Gustavo de Almeida, Dhoke, Gaurao V, Davari, Mehdi D, Ruff, Anna Joëlle, Schwaneberg, Ulrich
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Aqueous solutions; Bacillus megaterium - enzymology; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Benzene; Binding Sites; Biotransformation; Catalysis; Cloning; Cytochrome; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - metabolism; Directed evolution; E coli; Engineering; Enzyme Activation; Enzymes; Evolution; Gold; Heterocyclic Compounds - chemistry; High-throughput screening; Hydrocarbons; Hydroxylation; Models, Molecular; Molecular Conformation; Molecular dynamics; molecular modeling; Molecular Structure; monooxygenases aromatic heterocycles; Mutagenesis; Mutation; P450; Phenolic compounds; Phenols; Protein Binding; Protein Engineering; Standard deviation; Structure-Activity Relationship
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms20133353

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) 3 (BM3) wild type (WT) from 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 ; R255L: 1168 ± 163 min ), 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.