Nonribosomal peptide synthetases (NRPSs) are mega-enzymes responsible for the biosynthesis of many clinically important natural products, from early modern medicines (penicillin, bacitracin) to current blockbuster drugs (Cubicin, vancomycin) and newly-approved therapeutics (rezafungin) ^1,2. The key chemical step in these biosyntheses is amide bond formation between aminoacyl building blocks, catalyzed by the condensation (C) domain ³. There has been much debate over the mechanism of this reaction ^3-12. NRPS condensation has been difficult to fully characterize because it is one of many successive reactions in the NRPS synthetic cycle and because the canonical substrates are each attached transiently as thioesters to mobile carrier domains, which are often both contained in the same very flexible protein as the C domain. We have produced a dimodular NRPS protein in two parts, modified each with appropriate non-hydrolysable substrate analogs ^13,14 assembled the two parts with protein ligation ¹⁵, and solved structures of the substrate- and product-bound states. The structures show precise orientation of the megaenzyme preparing the nucleophilic attack of its key chemical step, and allow biochemical assays and quantum mechanical simulations to precisely interrogate the reaction. These data suggest that NRPSs C domains use a concerted reaction mechanism, where the active site histidine likely serves not a as general base, but as a crucial stabilizing hydrogen bond acceptor for the developing ammonium.

非核糖体肽合成酶 （NRPS） 是负责许多临床重要天然产物生物合成的巨型酶，从早期现代药物（青霉素、杆菌肽）到当前的畅销药物（Cubicin、万古霉素）和新批准的疗法（瑞扎芬净）^1,2。这些生物合成的关键化学步骤是在缩合 （C） 结构域 ³ 催化下，氨酰基结构单元之间形成酰胺键。关于这种反应的机制一直存在很多争论 ^3-12。NRPS 缩合很难完全表征，因为它是 NRPS 合成循环中许多连续反应之一，并且每个经典底物都以硫酯的形式瞬时连接到移动载体结构域，而这些结构域通常都包含在与 C 结构域相同的非常灵活的蛋白质中。我们生产了一种双侧 NRPS 蛋白，分为两部分，每部分用适当的不可水解底物类似物 ^13,14 修饰，用蛋白质连接组装两部分 ¹⁵，并解决了底物和产物结合状态的结构。这些结构显示了巨酶的精确方向，为关键化学步骤的亲核攻击做准备，并允许生化分析和量子力学模拟精确询问反应。这些数据表明 NRPSs C 结构域使用协同反应机制，其中活性位点组氨酸可能不作为一般基础，而是作为发育中的铵的关键稳定氢键受体。