美国加州大学Burkart Michael D团队报道了非核糖体肽生物合成中的交联模间缩合。相关研究成果发表在2024年12月11日出版的国际学术期刊《自然》。

非核糖体肽合成酶是用于生产关键治疗药物的流水线生物合成途径，通常被排列成称为巨型合成酶的大型多结构域蛋白质。人们使用肽基载体蛋白合成多肽，该蛋白通过模块化加载、修饰和延伸2步骤穿梭每个氨基酸，并且由于其多结构域和多模块架构的固有动力学，在结构表征方面仍然具有挑战性。

该文中，研究人员开发了位点选择性交联探针，以构象约束和解决载体蛋白与其伴侣酶结构域之间的相互作用。

研究人员应用四嗪点击化学将两种载体蛋白底物的缩合，捕获在缩合结构域的活性位点内，该缩合结构域将酪胺生物合成的前两个模块结合在一起，并报告了该复合物的高分辨冷冻EM结构。

与交联到其差向异构化结构域的第一个载体蛋白的X射线晶体结构一起，这些结构突出了捕获的互调识别事件，并定义了载体蛋白从一个催化步骤到下一个催化阶段的过程运动。

这些结构关系的表征仍然是理解这些独特合成酶分子细节的核心，并为未来这些途径的合成生物学设计提供了关键信息。

附：英文原文

Title: Crosslinking intermodular condensation in non-ribosomal peptide biosynthesis

Author: Heberlig, Graham W., La Clair, James J., Burkart, Michael D.

Issue&Volume: 2024-12-11

Abstract: Non-ribosomal peptide synthetases are assembly line biosynthetic pathways that are used to produce critical therapeutic drugs and are typically arranged as large multi-domain proteins called megasynthetases1. They synthesize polypeptides using peptidyl carrier proteins that shuttle each amino acid through modular loading, modification and elongation2 steps, and remain challenging to structurally characterize, owing in part to the inherent dynamics of their multi-domain and multi-modular architectures3. Here we have developed site-selective crosslinking probes to conformationally constrain and resolve the interactions between carrier proteins and their partner enzymatic domains4,5. We apply tetrazine click chemistry to trap the condensation of two carrier protein substrates within the active site of the condensation domain that unites the first two modules of tyrocidine biosynthesis and report the high-resolution cryo-EM structure of this complex. Together with the X-ray crystal structure of the first carrier protein crosslinked to its epimerization domain, these structures highlight captured intermodular recognition events and define the processive movement of a carrier protein from one catalytic step to the next. Characterization of these structural relationships remains central to understanding the molecular details of these unique synthetases and critically informs future synthetic biology design of these pathways.

DOI: 10.1038/s41586-024-08306-y

Source: https://www.nature.com/articles/s41586-024-08306-y