A Single Active Site Mutation in the Pikromycin Thioesterase Generates a More Effective Macrocyclization Catalyst

Macrolactonization of natural product analogs presents a significant challenge to both biosynthetic assembly and synthetic chemistry. In the preceding paper, we identified a thioesterase (TE) domain catalytic bottleneck processing unnatural substrates in the pikromycin (Pik) system, preventing the formation of epimerized macrolactones. Here, we perform molecular dynamics simulations showing the epimerized hexaketide was accommodated within the Pik TE active site; however, intrinsic conformational preferences of the substrate resulted in predominately unproductive conformations, in agreement with the observed hydrolysis. Accordingly, we engineered the stereoselective Pik TE to yield a variant (TES148C) with improved reaction kinetics and gain-of-function processing of an unnatural, epimerized hexaketide. Quantum mechanical comparison of model TES148C and TEWT reaction coordinate diagrams revealed a change in mechanism from a stepwise addition–elimination (TEWT) to a lower energy concerted acyl substitution (TES148C), accounting for the gain-of-function and improved reaction kinetics. Finally, we introduced the S148C mutation into a polyketide synthase module (PikAIII-TE) to impart increased substrate flexibility, enabling the production of diastereomeric macrolactones.

天然产物类似物的大环内酯化（macrolactonization）对生物合成组装（biosynthetic assembly）与合成化学均构成严峻挑战。在前期研究论文中，我们于匹克霉素（pikromycin, Pik）系统中发现，硫酯酶（thioesterase, TE）结构域在处理非天然底物时存在催化瓶颈，该瓶颈阻碍了差向异构大环内酯（epimerized macrolactones）的生成。本研究通过分子动力学模拟（molecular dynamics simulations）证实，差向异构六酮底物（epimerized hexaketide）可适配匹克霉素TE的活性位点（active site），但底物的固有构象偏好（intrinsic conformational preferences）使其主要呈现无效构象（unproductive conformations），这与实验观测到的水解反应（hydrolysis）结果一致。据此，我们对立体选择性匹克霉素TE实施工程化改造，获得突变体（TES148C）；该突变体不仅具备更优异的反应动力学（reaction kinetics）特性，还获得了功能增益（gain-of-function）活性，可处理非天然差向异构六酮底物。通过对TES148C与野生型TE（TEWT）的模型反应坐标图（reaction coordinate diagrams）开展量子力学（quantum mechanical）比较，我们发现其反应机制从野生型的分步加成-消除（stepwise addition–elimination）机制转变为能量更低的协同酰基取代（concerted acyl substitution）机制，这一变化解释了该突变体的功能增益与反应动力学提升现象。最后，我们将S148C突变引入聚酮合酶模块（polyketide synthase module, PikAIII-TE）中，使其底物柔性（substrate flexibility）显著增强，成功实现了非对映异构大环内酯（diastereomeric macrolactones）的制备。