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 (TE_S148C) with improved reaction kinetics and gain-of-function processing of an unnatural, epimerized hexaketide. Quantum mechanical comparison of model TE_S148C and TE_WT reaction coordinate diagrams revealed a change in mechanism from a stepwise addition–elimination (TE_WT) to a lower energy concerted acyl substitution (TE_S148C), 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.

中文翻译：

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吡咯霉素硫酯酶中的单个活性位点突变产生更有效的大环化催化剂

天然产物类似物的宏观内酯化对生物合成组装和合成化学都提出了重大挑战。在之前的文章中，我们确定了硫酯酶（TE）域催化瓶颈可处理吡咯霉素（Pik）系统中的非天然底物，从而防止了差向异构大内酯的形成。在这里，我们进行分子动力学模拟，表明差向异构的六酮化合物被容纳在Pik TE活性位点内。然而，与观察到的水解一致，底物的固有构象偏好导致主要是无用的构象。因此，我们设计了立体选择性Pik TE以产生变体（TE _S148C）具有改善的反应动力学和非天然，差向异构六酮化合物的功能获得过程。对模型TE _S148C和TE _WT反应坐标图的量子力学比较揭示了机理的变化，从逐步加成消除（TE _WT）到低能协同的酰基取代（TE _S148C），这说明了功能的获得并得到了改善反应动力学。最后，我们将S148C突变引入了聚酮化合物合酶模块（PikAIII-TE）中，以赋予底物更大的柔韧性，从而可以生产非对映体大内酯。