Our paper entitled “Molecular mechanism underlying substrate recognition of the peptide macrocyclase PsnB” in Nature Chemical Biology can be found here: https://www.nature.com/articles/s41589-021-00855-x

 Natural products have been a main source of therapeutic compounds. Among the various classes of natural products, ribosomally-synthesized and post-translationally modified peptides (RiPPs) have a distinct biosynthetic pathway, in which the ribosomally-produced precursor peptides undergo various chemical transformations by modification enzymes. The precursor peptides are often composed of two functional parts, a leader sequence for enzyme recognition and a core sequence for modification. This modular biosynthesis has been suggested to be the basis of high chemical diversity of RiPPs in natural evolution and high engineering potential for generating new functional peptides. The leader-mediated substrate recognition, however, significantly deters the determination of the core-bound enzyme structures, and thus, limits our understanding how enzymes recognize and modify the core region of the precursor peptides.

Biosynthesis of Plesiocin, a group 2 graspetide

 Our group has been interested in the chemical diversity of a family of RiPPs, graspetides or omega-ester containing peptides (OEPs), in which the class-defining modification generates the macrolactone or macrolactam linkages connecting two side chains. To understand the molecular mechanism of the macrolactone/macrolactam formation, we used the biosynthesis of plesiocin, a group 2 graspetide, as a model system to characterize the enzyme-substrate interactions and to obtain 3D structure of enzymes in complex with substrates. To our surprise, we found that the core region of the precursor peptide contributes to the enzyme-substrate interaction using the conserved ring-forming glutamate residues. The co-crystal structure of the enzyme-substrate complex not only confirmed the biochemical result, but revealed a distinct molecular mechanism for recognition of the ring-forming glutamate prior to macrocyclization; a highly-conserved arginine residue at the active site of the enzyme binds to the glutamate residue near the ATP-binding site.

 Molecular interaction of PsnB and core peptide

 Our structure is a rare example in which a large part of the core peptide is observed as a bound form in an RiPP biosynthetic enzyme. However, this study revealed only the first step of the macrocyclization reaction—recognition and placement of the ring-forming carboxyl acid before macrocyclization. Unanswered questions are: What residues in enzyme facilitate the next two steps of the macrocyclization—phosphorylation of the carboxyl acid and nucleophilic addition of a hydroxyl group? How are these two steps coordinated for overall macrocyclization reaction? What are the molecular basis of the enzyme specificity to the core sequence? Structures with compounds that mimic the reaction intermediates as well as detailed enzyme kinetic studies may provide additional information about the molecular mechanism of the macrocyclization. And the information obtained in this study will be a starting point to build graspetide-like peptide libraries for exploring diverse biological functions.