加拿大麦吉尔大学Schmeing, T. Martin团队报道了非核糖体肽合成中的缩合结构和机制。相关研究成果于2024年12月11日发表于国际一流学术期刊《自然》。

非核糖体肽合成酶（NRPSs）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）到目前的阻断药物（克必信、万古霉素）和新批准的治疗药物（雷扎芬金）。这些生物合成的关键化学步骤是由缩合（C）结构域催化的，氨基酰基构建块之间的酰胺键形成的。

关于这种反应的机制存在很多争论。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，并且因为典型底物各自以硫酯的形式，瞬时附着在移动载体结构域上，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。

研究人员分两部分制备了二聚体NRPS蛋白，用适当的不可水解底物类似物对每一部分进行修饰，用蛋白质连接组装两部分，并解决了底物结合态和产物结合态的结构。这些结构显示了巨酶的精确取向，为其关键化学步骤的亲核攻击做准备，并能够生化分析和量子力学模拟精确地询问反应。

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是作为一般碱基，而是作为发育铵的关键稳定氢键受体。

附：英文原文

Title: Structures and mechanism of condensation in nonribosomal peptide synthesis

Author: Pistofidis, Angelos, Ma, Pengchen, Li, Zihao, Munro, Kim, Houk, K. N., Schmeing, T. Martin

Issue&Volume: 2024-12-11

Abstract: 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.

DOI: 10.1038/s41586-024-08417-6

Source: https://www.nature.com/articles/s41586-024-08417-6