Molecular Evolution of an Aminotransferase Based on Substrate–Enzyme Binding Energy Analysis for Efficient Valienamine Synthesis
Li Cui, Anqi Cui, Qitong Li, Lezhou Yang, Hao Liu, Wenguang Shao, Yan Feng
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.