Mi Xuenan, Desormeaux Emily K, Le Tung T, van der Donk Wilfred A, Shukla Diwakar
Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign Urbana IL 61801 USA.
Department of Chemistry and Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign Urbana IL 61801 USA
Chem Sci. 2023 May 9;14(25):6904-6914. doi: 10.1039/d2sc06546k. eCollection 2023 Jun 28.
Lanthipeptides are ribosomally synthesized and post-translationally modified peptides that are generated from precursor peptides through a dehydration and cyclization process. ProcM, a class II lanthipeptide synthetase, demonstrates high substrate tolerance. It is enigmatic that a single enzyme can catalyze the cyclization process of many substrates with high fidelity. Previous studies suggested that the site-selectivity of lanthionine formation is determined by substrate sequence rather than by the enzyme. However, exactly how substrate sequence contributes to site-selective lanthipeptide biosynthesis is not clear. In this study, we performed molecular dynamic simulations for ProcA3.3 variants to explore how the predicted solution structure of the substrate without enzyme correlates to the final product formation. Our simulation results support a model in which the secondary structure of the core peptide is important for the final product's ring pattern for the substrates investigated. We also demonstrate that the dehydration step in the biosynthesis pathway does not influence the site-selectivity of ring formation. In addition, we performed simulation for ProcA1.1 and 2.8, which are well-suited candidates to investigate the connection between order of ring formation and solution structure. Simulation results indicate that in both cases, C-terminal ring formation is more likely which was supported by experimental results. Our findings indicate that the substrate sequence and its solution structure can be used to predict the site-selectivity and order of ring formation, and that secondary structure is a crucial factor influencing the site-selectivity. Taken together, these findings will facilitate our understanding of the lanthipeptide biosynthetic mechanism and accelerate bioengineering efforts for lanthipeptide-derived products.
羊毛硫肽是核糖体合成并经翻译后修饰的肽，通过脱水和环化过程从前体肽生成。ProcM是一种II类羊毛硫肽合成酶，具有高底物耐受性。令人费解的是，单一酶能够以高保真度催化多种底物的环化过程。先前的研究表明，羊毛硫氨酸形成的位点选择性由底物序列而非酶决定。然而，底物序列究竟如何促进位点选择性羊毛硫肽生物合成尚不清楚。在本研究中，我们对ProcA3.3变体进行了分子动力学模拟，以探索无酶情况下底物的预测溶液结构与最终产物形成之间的关联。我们的模拟结果支持一种模型，即核心肽的二级结构对于所研究底物的最终产物环模式很重要。我们还证明了生物合成途径中的脱水步骤不影响环形成的位点选择性。此外，我们对ProcA1.1和2.8进行了模拟，它们是研究环形成顺序与溶液结构之间联系的合适候选对象。模拟结果表明，在这两种情况下，C端环形成的可能性更大，这得到了实验结果的支持。我们的研究结果表明，底物序列及其溶液结构可用于预测环形成的位点选择性和顺序，并且二级结构是影响位点选择性的关键因素。综上所述，这些发现将有助于我们理解羊毛硫肽的生物合成机制，并加速羊毛硫肽衍生产品的生物工程研究。
