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The human gut microbiome significantly impacts the fate of ingested azo dyes primarily through the reduction of the azo bond (R\u2013N = N\u2013R\u2032). Flavin-dependent azoreductases have been considered the key enzymatic drivers of this process. To challenge this paradigm, we constructed a sequence similarity network of known azoreductases and focused on AzoA of Klebsiella pneumoniae, a central representative within the gut microbiome cluster. Unexpectedly, K. pneumoniae AzoA displayed relatively low in vitro activity against a range of azo compounds. While the physiological implications remain unclear, these findings are consistent with the possibility that nonenzymatic mechanisms contribute substantially to azo compound reduction in the gut. Furthermore, structural characterization of AzoA, as a holoenzyme and in its ligand-bound state, revealed a previously unrecognized ligand-coupled mechanism involving remodeling of the active site and oligomeric state.<\/jats:p>","DOI":"10.1021\/acs.biochem.6c00568","type":"journal-article","created":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T20:01:36Z","timestamp":1791403296000},"page":"3236-3246","source":"Crossref","is-referenced-by-count":0,"title":["Ligand Binding\nRemodels the Oligomeric State and Active\nSite of\n                    <i>Klebsiella pneumoniae<\/i>\n                    Azoreductase"],"prefix":"10.1021","volume":"65","author":[{"given":"Reese","family":"Hitchings","sequence":"first","affiliation":[{"name":"Albert Einstein College of Medicine , , , ,","place":["Bronx, New York, United States, 10461"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7753-0240","authenticated-orcid":true,"given":"Agnidipta","family":"Ghosh","sequence":"additional","affiliation":[{"name":"Albert Einstein College of Medicine , , , ,","place":["Bronx, New York, United States, 10461"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4903-3500","authenticated-orcid":true,"given":"Arthur J.","family":"Arcinas","sequence":"additional","affiliation":[{"name":"Albert Einstein College of Medicine , , , ,","place":["Bronx, New York, United States, 10461"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Steven C.","family":"Almo","sequence":"additional","affiliation":[{"name":"Albert Einstein College of Medicine , , , ,","place":["Bronx, New York, United States, 10461"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Libusha","family":"Kelly","sequence":"additional","affiliation":[{"name":"Department of Systems and Computational Biology, Department of Microbiology and Immunology Albert Einstein College of Medicine , , ,","place":["Bronx, New York, United States, 10461"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4627-8185","authenticated-orcid":true,"given":"Ali","family":"Ryan","sequence":"additional","affiliation":[{"name":"Northumbria University , , ,","place":["Newcastle-Upon-Tyne, United Kingdom, NE1 8ST"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,10,7]]},"reference":[{"key":"2026102007164485300_cit1","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1007\/978-3-319-33892-7_3","article-title":"Classification of dye and pigments","volume-title":"Dyes\nand Pigments","author":"Gu\u0308rses","year":"2016"},{"issue":"12","key":"2026102007164485300_cit2","doi-asserted-by":"publisher","first-page":"1113","DOI":"10.1177\/0009922816651621","article-title":"Prevalence of artificial\nfood colors in grocery store\nproducts marketed to children","volume":"55","author":"Batada","year":"2016","journal-title":"Clin. Pediatr."},{"key":"2026102007164485300_cit3","doi-asserted-by":"publisher","DOI":"10.1016\/j.fct.2023.113935","article-title":"Azo dyes in the food industry: Features,\nclassification, toxicity, alternatives, and regulation","volume":"178","author":"Barciela","year":"2023","journal-title":"Food Chem. Toxicol."},{"key":"2026102007164485300_cit4","doi-asserted-by":"publisher","DOI":"10.1016\/j.jfca.2022.104649","article-title":"Levels of FD&C\ncertified food dyes in foods commonly consumed by children","volume":"112","author":"Lehmkuhler","year":"2022","journal-title":"J. Food Compos. Anal."},{"issue":"4","key":"2026102007164485300_cit5","doi-asserted-by":"publisher","first-page":"309","DOI":"10.1177\/0009922814530803","article-title":"Amounts of artificial\nfood dyes and added sugars in foods and sweets commonly consumed by\nchildren","volume":"54","author":"Stevens","year":"2015","journal-title":"Clin. 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