Catalytic architecture and cyclase‐mediated dimerization of a newly identified class III ‐c lanthipeptide synthetase

Lanthipeptide synthetases catalyze dehydration and cyclization reactions that generate structurally complex ribosomally synthesized and post-translationally modified peptides. Class III LanKC enzymes are distinguished by their tri-functional kinase, lyase, and cyclase architecture, yet their mechanistic diversity remains incompletely understood due to limited structural and biochemical characterization. Here, we identify a previously unrecognized subgroup of Class III LanKC enzymes, designated Class III-c, through phylogenetic analysis and conserved substrate peptide features. Using cryo-electron microscopy, we determined the structure of a representative Class III-c enzyme, TherKC, in complex with its peptide substrate. We performed a systematic structural and functional characterization of TherKC, encompassing peptide substrate binding, oligomerization, and catalysis, providing a comprehensive mechanistic framework for this new LanKC class. TherKC assembles as a homodimer mediated exclusively by cyclase-cyclase interactions, a mode of oligomerization distinct from all previously characterized LanKC enzymes. Structural analysis identifies Arg685 as a conserved cyclase-domain hotspot that anchors dimer formation through symmetric electrostatic interactions. Disruption of this interface abolishes dimerization and reduces enzyme stability, while preserving substrate binding and catalytic competence. Structure-guided mutagenesis and mass spectrometric analyses further define conserved cyclase residues essential for efficient dehydration-cyclization coupling, provide insight into the formation of the conserved labionin-forming motif, revealing a noncanonical yet conserved catalytic framework. Together, these findings establish Class III-c LanKC enzymes as a mechanistically coherent subgroup and provide a comprehensive structural and functional understanding of this newly identified class, including its substrate-recognition, oligomerization, and cyclization mechanisms, thereby expanding the current landscape of lanthipeptide biosynthesis.