Chemical Probes Allow Structural Insight into the Condensation Reaction of Nonribosomal Peptide Synthetases
Chemical Probes Allow Structural Insight into the Condensation Reaction of Nonribosomal Peptide Synthetases
复制标题
DOI：
10.1016/j.chembiol.2016.02.012
复制
发表时间：
2016-03-17
影响因子：
8.6
通讯作者：
Schmeing, T. Martin
中科院分区：
文献类型：
--
作者：
Bloudoff, Kristjan;Alonzo, Diego A.;Schmeing, T. Martin
非核糖体肽合成酶（NRPS）合成大量的小分子，包括抗生素，抗肿瘤和免疫抑制剂。NRPS缩合（C）结构域催化酰胺键的形成，这是非核糖体肽合成的中心化学步骤。该反应的催化机理和底物决定因素仍在争论中。我们开发了化学探针来研究NRPS缩合反应的结构。这些底物类似物与活性位点附近引入的半胱氨酸共价连接，以模拟载体结构域的共价底物递送。它们是缩合反应中的感受态底物，其行为与天然底物相似。共晶结构显示C结构域-底物相互作用，并表明催化组氨酸的主要作用是定位α-氨基进行亲核攻击。由这些共复合物提供的结构洞察力也使我们能够用单个点突变改变反应的底物特异性概况。
Nonribosomal peptide synthetases (NRPSs) synthesize a vast variety of small molecules, including antibiotics, antitumors, and immunosuppressants. The NRPS condensation (C) domain catalyzes amide bond formation, the central chemical step in nonribosomal peptide synthesis. The catalytic mechanism and substrate determinants of the reaction are under debate. We developed chemical probes to structurally study the NRPS condensation reaction. These substrate analogs become covalently tethered to a cysteine introduced near the active site, to mimic covalent substrate delivery by carrier domains. They are competent substrates in the condensation reaction and behave similarly to native substrates. Co-crystal structures show C domain-substrate interactions, and suggest that the catalytic histidine's principle role is to position the a-amino group for nucleophilic attack. Structural insight provided by these co-complexes also allowed us to alter the substrate specificity profile of the reaction with a single point mutation.