Yu Du,Xiang Lv,Chenhao Feng,Yunjian Ma,Yonghua Wang
Enzymatic oxygenation of various cyclic ketones into lactones via Baeyer–Villiger monooxygenases (BVMOs) could provide a promising route for synthesizing fragrances and pharmaceutical ingredients. However, unsatisfactory catalytic activity and thermostability restricted their applications in the pharmaceutical and food industries. In this study, we successfully improved the catalytic activity and thermostability of a Baeyer–Villiger monooxygenase (OgBVMO) from Oceanicola granulosus by reshaping the binding pocket. As a result, mutant OgBVMO-Re displayed a 1.0- to 6.4-fold increase in the activity toward branched cyclic ketones tested, accompanied by a 3 °C higher melting point, and a 2-fold longer half-life time (t1/2 (45 °C)). Molecular dynamics simulations revealed that reshaping the binding pocket achieved strengthened motion correlation between amino acid residues, appropriate size of the substrate-binding pocket, beneficial surface characteristics, lower energy barriers, and shorter nucleophilic distance. This study well demonstrated the trade-off between the enzyme activity and thermostability by reshaping the substrate-binding pocket, paving the way for further engineering other enzymes.
Bin Ma,Jiafeng Niu,Hao Zhu,Huibing Chi,Zhaoxin Lu,Fengxia Lu,Ping Zhu
The oxidative reaction of Fusarium mycotoxin deoxynivalenol (DON) using the dehydrogenase is a desirable strategy and environmentally friendly to mitigate its toxicity. However, a critical issue for these dehydrogenases shows widespread substrate promiscuity. In this study, we conducted pocket reshaping of Devosia strain A6-243 pyrroloquinoline quinone (PQQ)-dependent dehydrogenase (DADH) on the basis of protein structure and kinetic analysis of substrate libraries to improve preference for particular substrate DON (10a). The variant presented an increased preference for substrate 10a and enhanced catalytic efficiency. A 4.7-fold increase in preference for substrate 10a was observed. Kinetic profiling and molecular dynamics (MD) simulations provided insights into the enhanced substrate specificity and activity. Moreover, the variant exhibited stronger conversion of substrate 10a to 3-keto-DON compared to the wild DADH. Overall, this study provides a feasible protocol for the redesign of PQQ-dependent dehydrogenases with favourable substrate specificity and catalytic activity, which is desperately needed for DON antidote development.
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