Abstract
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.
Highlights
Aromatic oxygen-containing heterocycles (O-heterocycles) are significantly abundant in nature as they are present in vitamins, hormones, antibiotics, sugars, pigments, and antioxidants and are involved in a variety of fundamental biological functions [1,2,3]
Cytochrome P450 monooxygenases (P450s) monooxygenase (P450) Bacillus megaterium 3 (BM3) wild type (WT) from Bacillus megaterium has low to no conversion of the six O-heterocycles
The latter led to the identification of position R255, which when introduced in the P450 BM3 WT, outperformed all other variants
Summary
Aromatic oxygen-containing heterocycles (O-heterocycles) are significantly abundant in nature as they are present in vitamins, hormones, antibiotics, sugars, pigments, and antioxidants (e.g., vitamin E, coumarin, flavonoids, and isoflavonoids) and are involved in a variety of fundamental biological functions [1,2,3]. Benzo-1,4-dioxane, a bicyclic heterocyclic compound consisting of a benzene ring fused to a heterocyclic dioxane ring, represents a series of synthetic and natural compounds [4,5,6,7,8,9,10,11] of considerable medicinal importance with various biological activities [12,13,14] such as antigrastic [15], spasmolytic [16], antipsychotic [17], anxiolytic [18], hepatoprotective [19], or α-adrenergic blocking agent activity [12,20,21] Functionalization of such heterocycles via chemical oxygenation is still challenging as it involves weary and costly steps that are catalyzed in the presence of expensive and toxic heavy metals [22,23] and often occur with little chemo-, regio-, and/or enantioselectivity leading to sustainability problems [23].
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