Promiscuous Enzymes Cooperate at the Substrate Level En Route to Lactazole A
Promiscuous Enzymes Cooperate at the Substrate Level En Route to Lactazole A
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DOI：
10.1021/jacs.0c05541
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发表时间：
2020-08-12
影响因子：
15
通讯作者：
Suga, Hiroaki
中科院分区：
文献类型：
--
作者：
Vinogradov, Alexander A.;Shimomura, Morito;Suga, Hiroaki
参与核糖体合成和翻译后修饰肽 (RiPP) 生物合成的酶通常具有宽松的特异性特征，并且能够修饰不同的底物。当几种这样的酶在前体肽成熟过程中一起作用时，可以形成多种产物，但通常生物合成集中在单一天然产物上。在大多数情况下，控制 RiPP 组装完整性的机制仍然难以捉摸。在这里，我们研究了乳唑 A 的生物合成，乳唑 A 是一种模型硫肽，由核糖体前体肽的五种混杂酶产生。使用我们的体外硫肽生产 (FIT-Laz) 系统，我们确定了个体修饰水平上生物合成事件的顺序，并通过参与酶的底物范围分析补充了这项研究。我们的结果揭示了一个不寻常但明确的组装过程，其中环脱水、脱氢丙氨酸形成和唑啉脱氢事件相互交织，这是由于每种乳唑酶的最小底物识别要求特征。此外，每种酶在指导 LazBF 介导的脱氢丙氨酸形成中发挥着作用，这是组装过程的中心主题。环脱水酶 LazDE 区分单个丝氨酸残基以形成唑啉，留下其余五个作为潜在的脱水酶底物。吡啶合酶 LazC 对 LazBF 施加动力学控制，以防止过度脱水硫肽的形成，而脱氢与脱氢丙氨酸装置的耦合则阻止脱水不足产物的生成。总而言之，我们的结果表明生物合成酶之间的底物水平合作维持了乳唑组装的完整性。这项工作增进了我们对 RiPP 生物合成过程的理解，并促进了硫肽生物工程。
Enzymes involved in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs) often have relaxed specificity profiles and are able to modify diverse substrates. When several such enzymes act together during precursor peptide maturation, a multitude of products can form, yet usually the biosynthesis converges on a single natural product. For the most part, the mechanisms controlling the integrity of RiPP assembly remain elusive. Here, we investigate the biosynthesis of lactazole A, a model thiopeptide produced by five promiscuous enzymes from a ribosomal precursor peptide. Using our in vitro thiopeptide production (FIT-Laz) system, we determine the order of biosynthetic events at the individual modification level and supplement this study with substrate scope analysis for participating enzymes. Our results reveal an unusual but well-defined assembly process where cyclodehydration, dehydroalanine formation, and azoline dehydrogenation events are intertwined due to minimal substrate recognition requirements characteristic of every lactazole enzyme. Additionally, each enzyme plays a role in directing LazBF-mediated dehydroalanine formation, which emerges as the central theme of the assembly process. Cyclodehydratase LazDE discriminates a single serine residue for azoline formation, leaving the remaining five as potential dehydratase substrates. Pyridine synthase LazC exerts kinetic control over LazBF to prevent the formation of overdehydrated thiopeptides, whereas the coupling of dehydrogenation to dehydroalanine installation impedes generation of underdehydrated products. Altogether, our results indicate that substrate-level cooperation between the biosynthetic enzymes maintains the integrity of lactazole assembly. This work advances our understanding of RiPP biosynthesis processes and facilitates thiopeptide bioengineering.