天然存在的羊毛硫肽,通过各种酶翻译后修饰的肽,作为抗生素有着巨大的希望。尽管进行了广泛的生化和结构研究,肽修饰之前的事件仍然知之甚少。这里,我们确定了具有不同结构和功能特征的羊毛硫氨酸合成酶KC(LanKC)酶的不同亚类。我们证明了PneKC,这个子类的成员,形成二聚体并具有GTP酶活性。通过PneKC的三个低温EM结构,我们说明了肽PneA结合的不同阶段,从初始识别到完全绑定。我们的结构显示了与PneA核心肽和GTPγS复合的激酶结构域,磷酸结合裂解酶结构域,和非常规的环化酶结构域。PneA的前导肽与门环相互作用,从延伸到螺旋构象的过渡。我们确定了一个二聚化热点,并提出了一种在紧张和放松构象之间切换酶的“负协同性”机制。此外,我们确定了环化酶域中一个重要的盐桥,与常规环化酶结构域中的不同。这些残基在LanKC亚类中是高度保守的,并且是两个特征基序的一部分。这些结果揭示了羊毛硫肽修饰酶组装的潜在差异,并加深了我们对这些多功能酶中变构的理解。
Naturally occurring lanthipeptides, peptides post-translationally modified by various enzymes, hold significant promise as antibiotics. Despite extensive biochemical and structural studies, the events preceding peptide modification remain poorly understood. Here, we identify a distinct subclass of lanthionine synthetase KC (LanKC) enzymes with distinct structural and functional characteristics. We show that PneKC, a member of this subclass, forms a dimer and possesses GTPase activity. Through three cryo-EM structures of PneKC, we illustrate different stages of peptide PneA binding, from initial recognition to full binding. Our structures show the kinase domain complexed with the PneA core peptide and GTPγS, a phosphate-bound lyase domain, and an unconventional cyclase domain. The leader peptide of PneA interact with a gate loop, transitioning from an extended to a helical conformation. We identify a dimerization hot spot and propose a \"negative cooperativity\" mechanism toggling the enzyme between tense and relaxed conformation. Additionally, we identify an important salt bridge in the cyclase domain, differing from those in in conventional cyclase domains. These residues are highly conserved in the LanKC subclass and are part of two signature motifs. These results unveil potential differences in lanthipeptide modification enzymes assembly and deepen our understanding of allostery in these multifunctional enzymes.