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.
Li Hanbing, Xu Chang, Xia Yilinting, Tang Zhixuan, Zhou Yongjun, Shi Ting
. Exploring the Biosynthesis of Cyclic Peptide Catalyzed by the PBP-Type Thioesterase Ulm16[J]. Journal of Shanghai Jiaotong University(Science), 2026
, 31(3)
: 537
-547
.
DOI: 10.1007/s12204-026-2928-7
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