Biochemistry. 2021 Feb 9;60(5):412-430. doi: 10.1021/acs.biochem.0c00919. Epub 2021 Jan 28.
Class II lanthipeptide synthetases (LanM enzymes) catalyze the multistep post-translational modification of genetically encoded precursor peptides into macrocyclic (often antimicrobial) lanthipeptides. The reaction sequence involves dehydration of serine/threonine residues, followed by intramolecular addition of cysteine thiols onto the nascent dehydration sites to construct thioether bridges. LanMs utilize two separate active sites in an iterative yet highly coordinated manner to maintain a remarkable level of regio- and stereochemical control over the multistep maturation. The mechanisms underlying this biosynthetic fidelity remain enigmatic. We recently demonstrated that proper function of the haloduracin β synthetase (HalM2) requires dynamic structural elements scattered across the surface of the enzyme. Here, we perform kinetic simulations, structural analysis of reaction intermediates, hydrogen-deuterium exchange mass spectrometry studies, and molecular dynamics simulations to investigate the contributions of these dynamic HalM2 structural elements to biosynthetic efficiency and fidelity. Our studies demonstrate that a large, conserved loop (HalM2 residues P349-P405) plays essential roles in defining the precursor peptide binding site, facilitating efficient peptide dehydration, and guiding the order of thioether ring formation. Moreover, mutations near the interface of the HalM2 dehydratase and cyclase domains perturb cyclization fidelity and result in aberrant thioether topologies that cannot be corrected by the wild type enzyme, suggesting an element of kinetic control in the normal cyclization sequence. Overall, this work provides the most comprehensive correlation of the structural and functional properties of a LanM enzyme reported to date and should inform mechanistic studies of the biosynthesis of other ribosomally synthesized and post-translationally modified peptide natural products.
II 类兰尼肽合成酶（LanM 酶）催化遗传编码的前体肽经多步翻译后修饰为大环（通常为抗微生物）兰尼肽。反应序列包括丝氨酸/苏氨酸残基的脱水，然后是半胱氨酸硫醇在新生脱水部位的分子内加成，以构建硫醚键。LanMs 以迭代但高度协调的方式利用两个独立的活性位点，对多步成熟过程保持着显著的区域和立体化学控制水平。这种生物合成保真度的机制仍然是个谜。我们最近证明，卤虫素β 合成酶（HalM2）的正常功能需要分散在酶表面的动态结构元件。在这里，我们进行了动力学模拟、反应中间体的结构分析、氢氘交换质谱研究和分子动力学模拟，以研究这些动态 HalM2 结构元件对生物合成效率和保真度的贡献。我们的研究表明，一个大的、保守的环（HalM2 残基 P349-P405）在确定前体肽结合位点、促进有效的肽脱水以及指导硫醚环形成的顺序方面起着至关重要的作用。此外，在 HalM2 脱水酶和环化酶结构域界面附近的突变会干扰环化保真度，并导致异常的硫醚拓扑结构，无法被野生型酶纠正，这表明在正常环化序列中存在动力学控制因素。总的来说，这项工作提供了迄今为止报道的 LanM 酶结构和功能特性的最全面相关性，应该为其他核糖体合成和翻译后修饰肽天然产物的生物合成的机制研究提供信息。
