Exploring the Substrate Stereoselectivity and Catalytic Mechanism of Nonribosomal Peptide Macrocyclization in Surugamides Biosynthesis.

SurE, the first reported penicillin -binding protein -like thioesterase (PBP-like TE), is known as a new offloading cyclase, which catalyzes heterochiral coupling in nonribosomal peptides (NRPs). However, the structural rationale for substrate stereoselectivity and enzymatic mechanism remains mysterious. Here, computational models, integrating MD simulations and QM/MM methods, unveiled SurE's substrate recognition and catalytic process. An oxyanion hole stabilized the C -terminal D -residue during recognition. Residue R446 anchored the substrate for macrocyclization. A vital hydrogen -bonding network (Y154, K66, N156), verified by mutation results, was responsible for the recognition of N -terminal L -residue and involvement in catalytic process with a calculated 19.4 kcal/mol energy barrier. Four novel -designed peptide precursors were effectively cyclized into cyclopeptides by SurE based on computational analysis. Our results provide a comprehensive understanding of SurE's catalytic mechanism and guiding design of versatile PBP-like TEs for novel macrocyclic NRPs.