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biological activity. However, the distribution of bacterial biosynthetic pathways that produce methylated cyclopropane amino acids and the structural basis of methyl-SAM recognition remain poorly understood. Here, we show that pyridoxal 5\u2032-phosphate (PLP)-dependent 1-amino-2-methylcyclopropane-1-carboxylic acid (2-methyl-ACC) synthases are widely distributed in bacteria. Although Orf30 preferentially converts (R)-4\u2033-methyl-SAM into (1S,2S)-methyl-ACC, some homologues also accept the (S)-4\u2033-methyl-SAM stereoisomer. Structural, computational, and mutational analyses of Orf30, the PLP-dependent enzyme responsible for formation of the 2-methyl-ACC moiety in the natural product Q6402A, suggest that the shape and hydrophobicity of the C4\u2033-methyl-binding region contribute to productive substrate positioning and stereochemical preference. Molecular dynamics simulations, density functional theory calculations, and mutational analyses further support direct cyclopropane formation from a PLP-bound quinonoid intermediate without requiring a substrate intermediate covalently bound to the enzyme at C4\u2033. Together, these findings show how active-site architecture couples substrate stereochemical recognition with quinonoid-driven cyclopropanation, expanding our understanding of ACC synthase-like PLP enzymes.<\/jats:p>","DOI":"10.1021\/jacs.6c13085","type":"journal-article","created":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T17:01:49Z","timestamp":1791478909000},"page":"44558-44566","source":"Crossref","is-referenced-by-count":0,"title":["Structural and Mechanistic\nBasis of Bacterial 2-Methyl-ACC\nSynthases"],"prefix":"10.1021","volume":"148","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5584-1822","authenticated-orcid":true,"given":"Lyu","family":"Zhou","sequence":"first","affiliation":[{"name":"The University of Tokyo , , ,","place":["Tokyo, Japan, 113-0033"]},{"name":"The University of Tokyo , , ,","place":["Tokyo, Japan, 113-8657"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2754-5858","authenticated-orcid":true,"given":"Takahiro","family":"Mori","sequence":"additional","affiliation":[{"name":"The University of Tokyo , , ,","place":["Tokyo, Japan, 113-0033"]},{"name":"The University of Tokyo , , ,","place":["Tokyo, Japan, 113-8657"]},{"name":"Japan Science and Technology Agency , , ,","place":["Saitama, Japan, 332-0012"]},{"name":"Waseda University , , ,","place":["Tokyo, Japan, 169-8555"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-1215-0168","authenticated-orcid":true,"given":"Takuto","family":"Ohmura","sequence":"additional","affiliation":[{"name":"The University of Tokyo , , ,","place":["Tokyo, Japan, 113-8657"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroyuki","family":"Seko","sequence":"additional","affiliation":[{"name":"The University of Tokyo , , ,","place":["Tokyo, Japan, 113-0033"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2575-3175","authenticated-orcid":true,"given":"Takayoshi","family":"Awakawa","sequence":"additional","affiliation":[{"name":"Center for Sustainable Resource Sciences , , ,","place":["Saitama, Japan, 351-0198"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4788-0063","authenticated-orcid":true,"given":"Fumitaka","family":"Kudo","sequence":"additional","affiliation":[{"name":"Kanagawa University , , ,","place":["Yokohama, Japan, 221-8686"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7091-0646","authenticated-orcid":true,"given":"Tohru","family":"Terada","sequence":"additional","affiliation":[{"name":"The University of Tokyo , , ,","place":["Tokyo, Japan, 113-8657"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3640-888X","authenticated-orcid":true,"given":"Ikuro","family":"Abe","sequence":"additional","affiliation":[{"name":"The University of Tokyo , , ,","place":["Tokyo, Japan, 113-0033"]},{"name":"The University of Tokyo , , ,","place":["Tokyo, Japan, 113-8657"]},{"name":"University of Toyama , , ,","place":["Toyama, Japan, 930-0194"]},{"name":"National Sun Yat-Sen University , , ,","place":["Kaohsiung, Taiwan, 80424"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,10,8]]},"reference":[{"issue":"6","key":"2026102111075861900_cit1","doi-asserted-by":"publisher","first-page":"892","DOI":"10.1104\/pp.60.6.892","article-title":"Methionine Metabolism in Apple Tissue: Implication\nof S-Adenosylmethionine as an Intermediate in the Conversion of Methionine\nto Ethylene","volume":"60","author":"Adams","year":"1977","journal-title":"Plant Physiol."},{"issue":"1","key":"2026102111075861900_cit2","doi-asserted-by":"publisher","first-page":"170","DOI":"10.1073\/pnas.76.1.170","article-title":"Ethylene biosynthesis:\nIdentification of 1-aminocyclopropane-1-carboxylic\nacid as an intermediate in the conversion of methionine to ethylene","volume":"76","author":"Adams","year":"1979","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"issue":"10","key":"2026102111075861900_cit3","doi-asserted-by":"publisher","first-page":"1063","DOI":"10.1038\/nchembio.2448","article-title":"Colibactin\nassembly line enzymes\nuse S-adenosylmethionine to build a cyclopropane ring","volume":"13","author":"Zha","year":"2017","journal-title":"Nat. Chem. Biol."},{"issue":"7054","key":"2026102111075861900_cit4","doi-asserted-by":"publisher","first-page":"1191","DOI":"10.1038\/nature03797","article-title":"Cryptic chlorination\nby a non-haem iron enzyme during cyclopropyl amino acid biosynthesis","volume":"436","author":"Vaillancourt","year":"2005","journal-title":"Nature"},{"issue":"38","key":"2026102111075861900_cit5","doi-asserted-by":"publisher","first-page":"6947","DOI":"10.1016\/S0040-4039(98)01460-9","article-title":"Biosynthesis\nof 1-aminocyclopropane-1-carboxylic\nacid moiety on cytotrienin A in Streptomyces sp","volume":"39","author":"Zhang","year":"1998","journal-title":"Tetrahedron Lett."},{"issue":"49","key":"2026102111075861900_cit6","doi-asserted-by":"publisher","first-page":"16957","DOI":"10.1021\/jacs.8b11463","article-title":"Discovery and Characterization of\n1-Aminocyclopropane-1-carboxylic Acid Synthase of Bacterial Origin","volume":"140","author":"Xu","year":"2018","journal-title":"J. Am. Chem. Soc."},{"issue":"5","key":"2026102111075861900_cit7","doi-asserted-by":"publisher","DOI":"10.3390\/biom10050775","article-title":"C-Methylation of S-adenosyl-L-Methionine\nOccurs Prior to Cyclopropanation in the Biosynthesis of 1-Amino-2-Methylcyclopropanecarboxylic\nAcid (Norcoronamic Acid) in a Bacterium","volume":"10","author":"Maruyama","year":"2020","journal-title":"Biomolecules"},{"issue":"26","key":"2026102111075861900_cit8","doi-asserted-by":"publisher","first-page":"9347","DOI":"10.1021\/ja902261a","article-title":"Escherichia coli Allows\nEfficient Modular Incorporation of Newly Isolated Quinomycin Biosynthetic\nEnzyme into Echinomycin Biosynthetic Pathway for Rational Design and\nSynthesis of Potent Antibiotic Unnatural Natural Product","volume":"131","author":"Watanabe","year":"2009","journal-title":"J. Am. Chem. Soc."},{"issue":"49","key":"2026102111075861900_cit9","doi-asserted-by":"publisher","first-page":"17719","DOI":"10.1021\/jacs.7b09971","article-title":"ClbS Is\na Cyclopropane Hydrolase\nThat Confers Colibactin Resistance","volume":"139","author":"Tripathi","year":"2017","journal-title":"J. Am. Chem.\nSoc."},{"issue":"5","key":"2026102111075861900_cit10","doi-asserted-by":"publisher","first-page":"411","DOI":"10.1038\/nchem.2221","article-title":"The colibactin warhead crosslinks DNA","volume":"7","author":"Vizcaino","year":"2015","journal-title":"Nat. Chem."},{"issue":"19","key":"2026102111075861900_cit11","doi-asserted-by":"publisher","first-page":"8712","DOI":"10.1021\/acs.jmedchem.6b00472","article-title":"The \u201cCyclopropyl\nFragment\u201d is a Versatile Player that Frequently Appears in\nPreclinical\/Clinical Drug Molecules","volume":"59","author":"Talele","year":"2016","journal-title":"J. Med.\nChem."},{"issue":"4","key":"2026102111075861900_cit12","doi-asserted-by":"publisher","first-page":"448","DOI":"10.1039\/D0MD00370K","article-title":"Put a ring\non it: application of small aliphatic rings in medicinal chemistry","volume":"12","author":"Bauer","year":"2021","journal-title":"RSC Med. Chem."},{"issue":"3","key":"2026102111075861900_cit13","doi-asserted-by":"publisher","first-page":"953","DOI":"10.1111\/j.1432-1033.1994.tb18577.x","article-title":"Evolutionary relationships\namong pyridoxal-5\u2032-phosphate-dependent enzymes","volume":"219","author":"Alexander","year":"1994","journal-title":"Eur. J. Biochem."},{"issue":"1","key":"2026102111075861900_cit14","doi-asserted-by":"publisher","first-page":"138","DOI":"10.1006\/bbrc.1994.1020","article-title":"Homology of 1-Aminocyclopropane-1-carboxylate\nSynthase,\n8-Amino-7-oxononanoate Synthase, 2-Amino-6-caprolactam Racemase, 2,2-Dialkylglycine\nDecarboxylase, Glutamate-1-semialdehyde 2,1-Aminomutase and Isopenicillin-N-Epimerase\nwith Aminotransferases","volume":"198","author":"Mehta","year":"1994","journal-title":"Biochem. Biophys. Res.\nCommun."},{"issue":"46","key":"2026102111075861900_cit15","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.abg8752","article-title":"Dual activities of ACC synthase:\nNovel clues regarding the molecular evolution of ACS genes","volume":"7","author":"Xu","year":"2021","journal-title":"Sci. Adv."},{"issue":"49","key":"2026102111075861900_cit16","doi-asserted-by":"publisher","first-page":"8975","DOI":"10.1021\/acs.orglett.2c03555","article-title":"Mechanism of S-Adenosyl-l-methionine C-Methylation\nby Cobalamin-dependent Radical S-Adenosyl-l-methionine Methylase in\n1-Amino-2-methylcyclopropanecarboxylic Acid Biosynthesis","volume":"24","author":"Kudo","year":"2022","journal-title":"Org. Lett."},{"key":"2026102111075861900_cit17","unstructured":"Kudo, F.\n          \n          C-Methylated natural\nproducts: biosynthetic stage and significance of C-methylation\n          Natural Product Reports\n          2026\n          10.1039\/D6NP00056H."},{"issue":"3","key":"2026102111075861900_cit18","doi-asserted-by":"publisher","first-page":"1681","DOI":"10.1021\/cr200073d","article-title":"Enzymatic Chemistry of Cyclopropane,\nEpoxide, and Aziridine Biosynthesis","volume":"112","author":"Thibodeaux","year":"2012","journal-title":"Chem. Rev."},{"issue":"418","key":"2026102111075861900_cit19","doi-asserted-by":"publisher","first-page":"2203","DOI":"10.1093\/jxb\/eri220","article-title":"Tyr152 plays\na central role in the\ncatalysis of 1-aminocyclopropane-1-carboxylate synthase","volume":"56","author":"Li","year":"2005","journal-title":"J. Exp. Bot."},{"issue":"41","key":"2026102111075861900_cit20","doi-asserted-by":"publisher","first-page":"38210","DOI":"10.1074\/jbc.M103840200","article-title":"Crystal Structures\nof 1-Aminocyclopropane-1-carboxylate (ACC) Synthase in Complex with\nAminoethoxyvinylglycine and Pyridoxal-5\u2032-Phosphate Provide\nNew Insight into Catalytic Mechanisms","volume":"276","author":"Huai","year":"2001","journal-title":"J. Biol.\nChem."},{"issue":"2","key":"2026102111075861900_cit21","doi-asserted-by":"publisher","first-page":"207","DOI":"10.1007\/BF01952472","article-title":"Biosynthesis of 1-aminocyclopropanecarboxylic\nacid: Steric course of the reaction at the C-4 position","volume":"42","author":"Wiesendanger","year":"1986","journal-title":"Experientia"},{"issue":"23","key":"2026102111075861900_cit22","doi-asserted-by":"publisher","first-page":"7820","DOI":"10.1073\/pnas.82.23.7820","article-title":"Stereochemical\ncourse of the reaction catalyzed by the pyridoxal phosphate-dependent\nenzyme 1-aminocyclopropane-1-carboxylate synthase","volume":"82","author":"Ramalingam","year":"1985","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"issue":"28","key":"2026102111075861900_cit23","doi-asserted-by":"publisher","first-page":"8754","DOI":"10.1002\/anie.201802479","article-title":"Biosynthesis and Heterologous Production\nof Vioprolides: Rational Biosynthetic Engineering and Unprecedented\n4-Methylazetidinecarboxylic Acid Formation","volume":"57","author":"Yan","year":"2018","journal-title":"Angew. Chem. Int. Ed"},{"issue":"1","key":"2026102111075861900_cit24","doi-asserted-by":"publisher","first-page":"99","DOI":"10.1021\/acschembio.8b00958","article-title":"Class I Methyltransferase\nVioH Catalyzes Unusual S-Adenosyl-l-methionine\nCyclization Leading to 4-Methylazetidinecarboxylic Acid Formation\nduring Vioprolide Biosynthesis","volume":"14","author":"Yan","year":"2019","journal-title":"ACS Chem. Biol."},{"issue":"1","key":"2026102111075861900_cit25","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-020-68860-z","article-title":"The molecular basis of thioalcohol\nproduction in human body odour","volume":"10","author":"Rudden","year":"2020","journal-title":"Sci. Rep."},{"issue":"3","key":"2026102111075861900_cit26","doi-asserted-by":"publisher","first-page":"745","DOI":"10.1006\/jmbi.1999.3255","article-title":"Structure\nof 1-aminocyclopropane-1-carboxylate synthase,\na key enzyme in the biosynthesis of the plant hormone ethylene","volume":"294","author":"Capitani","year":"1999","journal-title":"J. Mol. Biol."},{"issue":"1","key":"2026102111075861900_cit27","doi-asserted-by":"publisher","first-page":"279","DOI":"10.1016\/j.abb.2004.09.037","article-title":"Reaction\nspecificity in pyridoxal phosphate enzymes","volume":"433","author":"Toney","year":"2005","journal-title":"Arch. Biochem. Biophys."},{"issue":"11","key":"2026102111075861900_cit28","doi-asserted-by":"publisher","first-page":"1407","DOI":"10.1016\/j.bbapap.2011.05.019","article-title":"Controlling\nreaction specificity in pyridoxal phosphate enzymes","volume":"1814","author":"Toney","year":"2011","journal-title":"Biochim. Biophys. Acta, Proteins Proteomics"},{"issue":"6","key":"2026102111075861900_cit29","doi-asserted-by":"publisher","first-page":"476","DOI":"10.1038\/s41929-023-00963-y","article-title":"A pyridoxal\n5\u2032-phosphate-dependent Mannich cyclase","volume":"6","author":"Gao","year":"2023","journal-title":"Nat. Catal"},{"issue":"20","key":"2026102111075861900_cit30","doi-asserted-by":"publisher","first-page":"17191","DOI":"10.1021\/acscatal.5c03122","article-title":"Computational Investigation of the PazB-Catalyzed Cyclopropanation\nReaction: Role of Active-Site Water in SN2Mechanism","volume":"15","author":"Luo","year":"2025","journal-title":"ACS Catal."}],"container-title":["Journal of the American\nChemical Society"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/jacs.6c13085","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jacsat\/article-pdf\/148\/41\/44558\/68791210\/jacs.6c13085.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jacsat\/article-pdf\/148\/41\/44558\/68791210\/jacs.6c13085.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,21]],"date-time":"2026-10-21T15:31:55Z","timestamp":1792596715000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jacsat\/article\/148\/41\/44558\/5447208\/Structural-and-Mechanistic-Basis-of-Bacterial-2"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,10,8]]},"references-count":30,"journal-issue":{"issue":"41","published-online":{"date-parts":[[2026,10,8]]},"published-print":{"date-parts":[[2026,10,21]]}},"URL":"https:\/\/doi.org\/10.1021\/jacs.6c13085","relation":{},"ISSN":["0002-7863","1520-5126"],"issn-type":[{"value":"0002-7863","type":"print"},{"value":"1520-5126","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,10,8]]}}}