Abstract: Cyclopeptide compounds have emerged as a significant source of drug candidates in pharmaceutical development due to their unique cyclic structures, enhanced metabolic stability, and exceptional biological activities. Ulm16, a penicillin-binding protein-type thioesterase, promiscuously catalyzes the macrocyclization of linear peptides of different sizes between the N- and C-terminal residues with L- and D-configurations, respectively. However, the mechanism governing its selectivity towards substrates with various lengths remains unclear. This study comparatively investigated the structural differences in the interaction modes between Ulm16 and its linear substrates—hexapeptide (WLA-B1) and octapeptide (SGM)—by integrating molecular docking, molecular dynamics simulations, and rational design approaches. Findings revealed that the cooperative interactions with the oxygen anion cavity (Ser71/Thr299) and Arg431 in the active pockets stabilize WLA-B1 at both the C- and N-termini, which is crucial for its efficient macrocyclization. Conversely, SGM exhibits impaired pre-reaction state proportion due to the absence of key hydrogen-bonding networks. Based on structural analysis, we designed a series of mutants (A225S, G226E, D147V, S144F, and L300G) to optimize substrate binding by enhancing the negative charge density within the active pocket. Among them, A225S variant demonstrated a slight improvement in the yield of cyclized WLA-B1 (1.3-fold higher than the wild type). This study established a computational methodology spanning from substrate loading to catalytic macrocyclization, elucidated the molecular mechanism of substrate selectivity with Ulm16, and provided crucial theoretical foundations for rationally designing highly efficient cyclic peptide biosynthetic enzymes.

Key words: cyclic peptide, penicillin-binding protein, thioesterase, molecular mechanism, rational engineering

摘要： 环肽类化合物因其独特的环状结构与优良的生物活性，已成为药物研发领域重要的候选药物来源。Ulm16是一种青霉素结合蛋白型（PBP型）硫酯酶，能够催化多种线性肽在N端与C端残基之间发生大环化反应。然而，其识别选择性机制尚不明确。本研究通过整合分子对接、分子动力学模拟及理性设计等方法，比较分析了Ulm16与其线性六肽底物（WLA-B1）和八肽底物（SGM）之间相互作用模式的结构差异。研究表明，Ulm16活性口袋中氧阴离子穴（Ser71/Thr299）与Arg431的协同相互作用能够稳定六肽底物WLA-B1的C端和N端，这对于其高效发生大环化反应至关重要。相比之下，八肽底物SGM由于缺乏关键的氢键网络，其预反应状态的占比降低。基于结构分析，我们理性设计了一系列突变体（A225S/G226E/D147V/S144F/L300G）以优化底物结合。其中，A225S突变体环化六肽底物WLA-B1的产率较野生型提高了1.3倍。本研究建立了从底物加载到催化大环化的计算方法，阐明了Ulm16底物选择性的分子机制，并为理性设计高效环肽生物合成酶提供了重要的理论依据。

关键词: 环肽，青霉素结合蛋白，硫酯酶，分子机制，理性改造