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This pyrrolobenzodiazepine (PBD) natural product is synthesized by a bimodular nonribosomal peptide synthetase (NRPS) pathway composed of three proteins: NpsA, ThdA, and NpsB. We describe the functional and structural characterization of the fully reconstituted NRPS system and report the steady-state kinetic analysis of all natural substrates and cofactors as well as the structural characterization of both NpsA and ThdA. The mechanism of action of tilimycin was confirmed using DNA adductomics techniques through the detection of putative N-2 guanine alkylation after tilimycin exposure to eukaryotic cells, providing the first structural characterization of a PBD\u2013DNA adduct formed in cells. Finally, we report the rational design of small-molecule inhibitors that block tilimycin biosynthesis in whole cell K.\u00a0oxytoca (IC50 = 29 \u00b1 4 \u03bcM) through the inhibition of NpsA (KD = 29 \u00b1 4 nM).<\/jats:p>","DOI":"10.1021\/acsinfecdis.0c00326","type":"journal-article","created":{"date-parts":[[2020,6,2]],"date-time":"2020-06-02T17:47:43Z","timestamp":1591120063000},"page":"1976-1997","source":"Crossref","is-referenced-by-count":39,"title":["Biosynthesis, Mechanism of Action, and Inhibition\nof the Enterotoxin Tilimycin Produced by the Opportunistic Pathogen\n                    <i>Klebsiella oxytoca<\/i>"],"prefix":"10.1021","volume":"6","author":[{"given":"Evan M.","family":"Alexander","sequence":"first","affiliation":[{"name":"University of Minnesota , , , ,","place":["Minneapolis, Minnesota, United States, 55455"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4758-7913","authenticated-orcid":true,"given":"Dale F.","family":"Kreitler","sequence":"additional","affiliation":[{"name":"University at Buffalo , , , ,","place":["Buffalo, New York, United States, 14203"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Valeria","family":"Guidolin","sequence":"additional","affiliation":[{"name":"University of Minnesota , , , ,","place":["Minneapolis, Minnesota, United States, 55455"]},{"name":"University of Minnesota , , , ,","place":["Minneapolis, Minnesota, United States, 55455"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexander K.","family":"Hurben","sequence":"additional","affiliation":[{"name":"University of Minnesota , , , ,","place":["Minneapolis, Minnesota, United States, 55455"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eric","family":"Drake","sequence":"additional","affiliation":[{"name":"University at Buffalo , , , ,","place":["Buffalo, New York, United States, 14203"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0067-3083","authenticated-orcid":true,"given":"Peter W.","family":"Villalta","sequence":"additional","affiliation":[{"name":"University of Minnesota , , , ,","place":["Minneapolis, Minnesota, United States, 55455"]},{"name":"University of Minnesota , , , ,","place":["Minneapolis, Minnesota, United States, 55455"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7686-0504","authenticated-orcid":true,"given":"Silvia","family":"Balbo","sequence":"additional","affiliation":[{"name":"University of Minnesota , , , ,","place":["Minneapolis, Minnesota, United States, 55455"]},{"name":"University of Minnesota , , , ,","place":["Minneapolis, Minnesota, United States, 55455"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4238-7453","authenticated-orcid":true,"given":"Andrew M.","family":"Gulick","sequence":"additional","affiliation":[{"name":"University at Buffalo , , , ,","place":["Buffalo, New York, United States, 14203"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9261-594X","authenticated-orcid":true,"given":"Courtney C.","family":"Aldrich","sequence":"additional","affiliation":[{"name":"University of Minnesota , , , ,","place":["Minneapolis, Minnesota, United States, 55455"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2020,6,2]]},"reference":[{"key":"2026090313171473900_cit1","doi-asserted-by":"publisher","first-page":"107","DOI":"10.1146\/annurev.mi.31.100177.000543","article-title":"Microbial\nEcology of the Gastrointestinal Tract","volume":"31","author":"Savage","year":"1977","journal-title":"Annu.\nRev. 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