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• W4307392010 abstract "Valienamine is a valuable building block for active pharmaceuticals and agrochemicals because of its aminocyclitol structure and glucosidase inhibitory activity. Straightforward amino chiral center construction on valienone using a sugar aminotransferase (SAT) to produce valienamine achieved strict stereo-specificity; however, low transamination activity owing to an unfavorable binding conformation of the non-natural substrate valienone in the oversized substrate-binding pocket currently limits SAT-based valienamine production. Here, we employed a tailored combinatorial active-site saturation test/iterative saturation mutagenesis (CAST/ISM) strategy to engineer a recently identified SAT (RffA_Kpn) to optimize the binding of valienone in the large binding pocket and thus improve transamination activity. In silico analyses predicting mutation binding energies and assessing evolutionary conservation identified 9 of 62 contact residues positioned within 8 Å of the valienone–PMP external aldimine transition state as hotspots for destabilizing unfavorable binding. Four of these residues were subsequently confirmed to improve activity using site-directed saturation mutagenesis, and combinatorial mutations were prepared in further iterative cycles. The quadruple mutant M4 (K209W/Y321F/V318Q/K25W) displayed a 35.59-fold improvement in valienamine synthesis activity over wild-type RffA_Kpn and achieved 12.18% conversion toward valienone in 100 mg preparative-scale reaction. Our demonstration of asymmetric biosynthesis for a valuable chiral aminocyclitol illustrates how an evolution model can help engineer enzymes that handle small, non-natural substrates in oversized binding pockets." @default.
• W4307392010 created "2022-11-01" @default.
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• W4307392010 date "2022-10-25" @default.
• W4307392010 modified "2023-10-17" @default.
• W4307392010 title "Molecular Evolution of an Aminotransferase Based on Substrate–Enzyme Binding Energy Analysis for Efficient Valienamine Synthesis" @default.
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• W4307392010 doi "https://doi.org/10.1021/acscatal.2c03784" @default.
• W4307392010 hasPublicationYear "2022" @default.
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