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HACL\/S natively cleaves 2-hydroxyacyl-CoAs and, in its reverse direction, condenses formyl-CoA with aldehydes or ketones. The one-carbon elongation biochemistry based on HACL\/S has enabled the use of molecules derived from greenhouse gases as biomanufacturing feedstocks. We investigated several HACL\/S family members with high activity in the condensation of formyl-CoA and aldehydes, and distinct chain-length specificities and kinetic parameters. Our analysis revealed the structures of enzymes in complex with acyl-CoA substrates and products, several covalent intermediates, bound ThDP and ADP, as well as the C-terminal active site region. One of these observed states corresponds to the intermediary \u03b1\u2013carbanion with hydroxymethyl-CoA covalently attached to ThDP. This research distinguishes HACL\/S from related sub-families and identifies key residues involved in substrate binding and catalysis. These findings expand our knowledge of acyloin-condensation biochemistry and offer attractive prospects for biocatalysis using carbon elongation.<\/jats:p>","DOI":"10.1038\/s42004-024-01242-y","type":"journal-article","created":{"date-parts":[[2024,7,21]],"date-time":"2024-07-21T09:01:33Z","timestamp":1721552493000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Revealing reaction intermediates in one-carbon elongation by thiamine diphosphate\/CoA-dependent enzyme family"],"prefix":"10.1038","volume":"7","author":[{"given":"Youngchang","family":"Kim","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Seung Hwan","family":"Lee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Priyanka","family":"Gade","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maren","family":"Nattermann","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2871-465X","authenticated-orcid":false,"given":"Natalia","family":"Maltseva","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Endres","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2620-360X","authenticated-orcid":false,"given":"Philipp","family":"Wichmann","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yang","family":"Hu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-1314-4583","authenticated-orcid":false,"given":"Daniel G.","family":"Marchal","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8372-640X","authenticated-orcid":false,"given":"Yasuo","family":"Yoshikuni","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3685-0894","authenticated-orcid":false,"given":"Tobias J.","family":"Erb","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4797-6580","authenticated-orcid":false,"given":"Ramon","family":"Gonzalez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7140-3649","authenticated-orcid":false,"given":"Karolina","family":"Michalska","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2535-6209","authenticated-orcid":false,"given":"Andrzej","family":"Joachimiak","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,7,21]]},"reference":[{"key":"1242_CR1","doi-asserted-by":"publisher","DOI":"10.1016\/j.sbi.2022.102441","volume":"76","author":"S Prajapati","year":"2022","unstructured":"Prajapati, S., Rabe von Pappenheim, F. & Tittmann, K. 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