State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.
Appl Microbiol Biotechnol. 2020 Jan;104(2):653-660. doi: 10.1007/s00253-019-10251-0. Epub 2019 Dec 4.
Lipopeptides are important non-ribosomal peptide synthetases (NRPSs) products with broad therapeutic potential in biotechnology and biopharmaceutical applications. Fatty acyl modifications in N-terminal of lipopeptides have attracted wide interest in the engineering processes of altered fatty acyl selectivity. In this study, we focused on the starter condensation domain of antibiotic A54145 (lptC1) and its indiscriminate selectivity of fatty acyl substrates, which results in multi-component products. Using in silico analysis, five site-directed mutations at protein-protein interface were identified with altered activity and selectivity towards wild type lptC1. The variants Y149W and A330T exhibited changed substrate selectivity to prefer longer branched chain fatty acyl substrate, while T16A and A350D showed improved selectivity for shorter linear chain fatty acyl substrates. Subsequently, molecular dynamics (MD) simulations were performed to analyze the impact of these residues on the changes of catalytic activity and conformation. Through in silico analysis, the altered binding free energy were coincident with the corresponding activity performance of the variants, and surface forces indicated that other factors or processes may influence the activity and selectivity. Moreover, the MD results revealed even altered active center conformations, implying the importance of these interface residues affected on distant active center thus reflected to catalysis activity. Based on the biochemistry and computational results, our work provides detailed insights from molecular and dynamics aspects into the role of C1's interface residues during complex NRPS biosynthesis machinery, prompting further rational engineering for lipopeptide catalysis.
脂肽是具有广泛治疗潜力的非核糖体肽合成酶（NRPSs）产物，在生物技术和生物制药应用中有重要作用。脂肽 N 端的脂肪酸酰基修饰在改变脂肪酸酰基选择性的工程化过程中引起了广泛关注。在这项研究中，我们专注于抗生素 A54145（lptC1）起始缩合结构域及其对脂肪酸酰基底物的无差别选择性，这导致了多组分产物的产生。通过计算机分析，在蛋白质-蛋白质界面处确定了五个定点突变，这些突变改变了野生型 lptC1 的活性和对脂肪酸酰基底物的选择性。变体 Y149W 和 A330T 表现出对更长支链脂肪酸酰基底物的改变的底物选择性，而 T16A 和 A350D 对更短的线性链脂肪酸酰基底物表现出改善的选择性。随后，进行了分子动力学（MD）模拟，以分析这些残基对催化活性和构象变化的影响。通过计算机分析，改变的结合自由能与变体的相应活性表现一致，表面力表明其他因素或过程可能会影响活性和选择性。此外，MD 结果表明甚至改变了活性中心构象，这意味着这些界面残基对遥远的活性中心的影响，从而反映了对催化活性的重要性。基于生物化学和计算结果，我们的工作从分子和动力学方面提供了对 C1 界面残基在复杂 NRPS 生物合成机制中的作用的详细见解，为脂肽催化的进一步合理工程提供了启示。
