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Class III LanKC enzymes are distinguished by their tri\u2010functional 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\u2010c, through phylogenetic analysis and conserved substrate peptide features. Using cryo\u2010electron microscopy, we determined the structure of a representative Class III\u2010c 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\u2013cyclase interactions, a mode of oligomerization distinct from all previously characterized LanKC enzymes. Structural analysis identifies Arg685 as a conserved cyclase\u2010domain 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\u2010guided mutagenesis and mass spectrometric analyses further define conserved cyclase residues essential for efficient dehydration\u2013cyclization coupling, provide insight into the formation of the conserved labionin\u2010forming motif, revealing a noncanonical yet conserved catalytic framework. Together, these findings establish Class III\u2010c LanKC enzymes as a mechanistically coherent subgroup and provide a comprehensive structural and functional understanding of this newly identified class, including its substrate\u2010recognition, oligomerization, and cyclization mechanisms, thereby expanding the current landscape of lanthipeptide biosynthesis.<\/jats:p>","DOI":"10.1002\/pro.70776","type":"journal-article","created":{"date-parts":[[2026,8,25]],"date-time":"2026-08-25T05:59:46Z","timestamp":1787637586000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Catalytic architecture and cyclase\u2010mediated dimerization of a newly identified class\n                    <scp>III<\/scp>\n                    \u2010c lanthipeptide synthetase"],"prefix":"10.1002","volume":"35","author":[{"given":"Haojie","family":"Zhang","sequence":"first","affiliation":[{"name":"Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering Chongqing University  Chongqing China"},{"name":"Department of Biological Sciences, Faculty of Science National University of Singapore  Singapore Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1103-7701","authenticated-orcid":false,"given":"Yifan","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, Faculty of Science National University of Singapore  Singapore Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yi","family":"Shi","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, Faculty of Science National University of Singapore  Singapore Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jian","family":"Shi","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, Faculty of Science National University of Singapore  Singapore Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bochu","family":"Wang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering Chongqing University  Chongqing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3493-2282","authenticated-orcid":false,"given":"Min","family":"Luo","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, Faculty of Science National University of Singapore  Singapore Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,8,24]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-024-07487-w"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.1039\/C2NP20085F"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1039\/D5NP00042D"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.2174\/138920109787048616"},{"key":"e_1_2_10_6_1","doi-asserted-by":"publisher","DOI":"10.1107\/S2059798320009080"},{"key":"e_1_2_10_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/nchembio.1856"},{"key":"e_1_2_10_8_1","doi-asserted-by":"publisher","DOI":"10.1021\/acschemneuro.8b00003"},{"key":"e_1_2_10_9_1","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.07607"},{"key":"e_1_2_10_10_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-024-02373-9"},{"key":"e_1_2_10_11_1","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.2c00727"},{"key":"e_1_2_10_12_1","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444911047834"},{"key":"e_1_2_10_13_1","doi-asserted-by":"publisher","DOI":"10.1039\/D0CB00073F"},{"key":"e_1_2_10_14_1","doi-asserted-by":"publisher","DOI":"10.1021\/acscentsci.3c00484"},{"key":"e_1_2_10_15_1","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202211382"},{"key":"e_1_2_10_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.foodres.2023.113208"},{"key":"e_1_2_10_17_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-023-01524-6"},{"key":"e_1_2_10_18_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-biochem-060110-113521"},{"key":"e_1_2_10_19_1","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.4c00266"},{"key":"e_1_2_10_20_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2007.05.022"},{"key":"e_1_2_10_21_1","doi-asserted-by":"publisher","DOI":"10.1007\/s11033-024-09582-7"},{"key":"e_1_2_10_22_1","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2020.01183"},{"key":"e_1_2_10_23_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-biochem-032620-104956"},{"key":"e_1_2_10_24_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1121422"},{"key":"e_1_2_10_25_1","doi-asserted-by":"publisher","DOI":"10.3389\/fbioe.2021.692466"},{"key":"e_1_2_10_26_1","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.5c00378"},{"key":"e_1_2_10_27_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-51600-6"},{"key":"e_1_2_10_28_1","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.2c00458"},{"key":"e_1_2_10_29_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-clinpsy-080822-051727"},{"key":"e_1_2_10_30_1","doi-asserted-by":"publisher","DOI":"10.1006\/abio.1994.1571"},{"key":"e_1_2_10_31_1","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2007.321"},{"key":"e_1_2_10_32_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature13888"},{"key":"e_1_2_10_33_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jsb.2023.107939"},{"key":"e_1_2_10_34_1","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.2c06808"},{"key":"e_1_2_10_35_1","doi-asserted-by":"publisher","DOI":"10.1002\/pro.4992"},{"key":"e_1_2_10_36_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.4169"},{"key":"e_1_2_10_37_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-020-00990-8"},{"key":"e_1_2_10_38_1","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.0c00127"},{"key":"e_1_2_10_39_1","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.4c00637"},{"key":"e_1_2_10_40_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrev.6b00591"},{"key":"e_1_2_10_41_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gku316"},{"key":"e_1_2_10_42_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.micro.52.1.41"},{"key":"e_1_2_10_43_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkm067"},{"key":"e_1_2_10_44_1","article-title":"NCBI 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