Asymmetric epoxidation is a green route to enantiopure epoxides, but often suffers from low enantioselectivity toward unconjugated terminal alkenes. Mining of the NCBI non-redundant protein sequences with a reconstructed ancestral sequence based on six styrene monooxygenases identified a monooxygenase (HhMo) from Herbaspirillum huttiense with 29.6–32.3% sequence identity with styrene monooxygenases, which was previously annotated as an alanine phosphoribitol ligase. HhMo catalyzed the epoxidation of allylbenzenes with moderate to excellent enantioselectivity yielding the corresponding epoxides in up to 99% ee. The HhMo-catalyzed epoxidation could also achieve the kinetic resolution of racemic secondary allylic alcohols, yielding the corresponding epoxides with up to 50% yields, as well as excellent enantio- and diastereoselectivity (up to >99% ee and >99% de) within 20–60 min, making a greener strategy for the production of valuable enantiopure glycidol derivatives in the fine chemical and pharmaceutical industries.
Abstract Styrene monooxygenases (SMOs) are powerful enzymes for the synthesis of enantiopure epoxides, but these SMOs have narrow substrate spectra, and the residues in controlling enantioselectivity of SMOs remains unclear. A monooxygenase from Herbaspirillum huttiense ( Hh MO) was found to have excellent enantioselectivities and diastereoselectivities in the epoxidation of unconjugated terminal alkenes. Here we found that Hh MO could also transfer styrene into styrene epoxide with 75% ee, and it could also catalyze the epoxidation of styrene derivatives into the corresponding epoxides with enantioselectivities up to 99% ee. Meanwhile, site 199 in the substrate access channel of Hh MO was found to play an important role in the controlling enantioselectivity of the epoxidation. The E199L variant catalyzed the epoxidation of styrene with > 99% ee. The identification of critical residue that affects the enantioselectivity of SMOs would thus be valuable for creating efficient monooxygenases for the preparation of essential enantiopure epoxides. Key points
Bioexpoxidation of both conjugated and unconjugated alkenes by HhMO with excellent enantioselectivities.
Gating residue 199 played an essential role in controlling the enantioselectivity of SMO.
HhMO E199L catalyzed the epoxidation of styrenes with up to > 99% ee.
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