Molecular Insights into Bifunctional Ambruticin DH3 for Substrate Specificity and Catalytic Mechanism.

Dehydratase (DH), a domain located at polyketide synthase (PKS) modules, commonly catalyzes the dehydration of ß-hydroxy to an α,ß-unsaturated acyl intermediate. As a unique bifunctional dehydratase, AmbDH3 (the DH domain of module 3 of the ambruticin PKS) is verified to be responsible for both dehydration and the following pyran-forming cyclization. Besides, in vitro studies showed that its catalytic efficiency varies with different chiral substrates. However, the detailed molecular mechanism of AmbDH3 remains unclear. In this work, the structural rationale for the substrate specificity (2R/2S- and 6R/6S-substrates) in AmbDH3 was elucidated and the complete reaction pathways including dehydration and cyclization were presented. Both MD simulations and binding free energy calculations indicated AmbDH3 had a stronger preference for 2R-substrates (2R6R-2, 2R6S-3) than 2S-substrates (2S6R-1), and residue H51 and G61 around the catalytic pocket were emphasized by forming stable hydrogen bonds with 2R-substrates. In addition, AmbDH3's mild tolerance at C6 was explained by comparison of substrate conformation and hydrogen bond network in 6S- and 6R-substrate systems. The QM/MM results supported a consecutive one-base dehydration and cyclization mechanism for 2R6S-3 substrate with the energy barrier of 25.2 kcal mol-1 and 24.5 kcal mol-1 , respectively. Our computational results uncover the substrate recognition and catalytic process of the first bifunctional dehydratase-cyclase AmbDH3, which will shed light on the application of multifunctional DH domains in PKSs for diverse natural product analogs and benefit the chemoenzymatic synthesis of stereoselective pyran-containing products.

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 off-loading 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.

Computer-aided molecular design and optimization of potent inhibitors disrupting APC‒Asef interaction.

Colorectal cancer (CRC) is the second leading cause of cancer mortality worldwide. At initial diagnosis, approximately 20% of patients are diagnosed with metastatic CRC (mCRC). Although the APC‒Asef interaction is a well-established target for mCRC therapy, the discovery and development of effective and safe drugs for mCRC patients remains an urgent and challenging endeavor. In this study, we identified a novel structural scaffold based on MAI inhibitors, the first-in-class APC‒Asef inhibitors we reported previously. ONIOM model-driven optimizations of the N-terminal cap and experimental evaluations of inhibitory activity were performed, and 24-fold greater potency was obtained with the best inhibitor compared to the parental compound. In addition, the cocrystal structure validated that the two-layer π‒π stacking interactions were essential for inhibitor stabilization in the bound state. Furthermore, in vitro and in vivo studies have demonstrated that novel inhibitors suppressed lung metastasis in CRC by disrupting the APC‒Asef interaction. These results provide an intrinsic structural basis to further explore drug-like molecules for APC‒Asef-mediated CRC therapy.

C-N bond formation by a polyketide synthase.

Assembly-line polyketide synthases (PKSs) are molecular factories that produce diverse metabolites with wide-ranging biological activities. PKSs usually work by constructing and modifying the polyketide backbone successively. Here, we present the cryo-EM structure of CalA3, a chain release PKS module without an ACP domain, and its structures with amidation or hydrolysis products. The domain organization reveals a unique "∞"-shaped dimeric architecture with five connected domains. The catalytic region tightly contacts the structural region, resulting in two stabilized chambers with nearly perfect symmetry while the N-terminal docking domain is flexible. The structures of the ketosynthase (KS) domain illustrate how the conserved key residues that canonically catalyze C-C bond formation can be tweaked to mediate C-N bond formation, revealing the engineering adaptability of assembly-line polyketide synthases for the production of novel pharmaceutical agents.