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Our biochemical assays characterize vcFabH1 as exhibiting specificity for acetyl\u2010CoA and CoA thioesters with short acyl chains, similar to that observed for FabH homologs found in most gram\u2010negative bacteria. vcFabH2 prefers medium chain\u2010length acyl\u2010CoA thioesters, particularly octanoyl\u2010CoA, which is a pattern of specificity rarely seen in bacteria. Structural characterization of one vcFabH1 and six vcFabH2 structures determined in either apo form or in complex with acetyl\u2010CoA\/octanoyl\u2010CoA indicate that the substrate\u2010binding pockets of vcFabH1 and vcFabH2 are of different sizes, accounting for variations in substrate chain\u2010length specificity. An unusual and unique feature of vcFabH2 is its C\u2010terminal fragment that interacts with both the substrate\u2010entrance loop and the dimer interface of the enzyme. Our discovery of the pattern of substrate specificity of both vcFabH1 and vcFabH2 can potentially aid the development of novel antibacterial agents against\n                    <jats:italic>V.\u00a0cholerae<\/jats:italic>\n                    . Additionally, the distinctive substrate preference of FabH2 in\n                    <jats:italic>V.\u00a0cholerae<\/jats:italic>\n                    and related facultative anaerobes conceivably make it an attractive component of genetically engineered bacteria used for commercial biofuel production.\n                  <\/jats:p>","DOI":"10.1111\/febs.14588","type":"journal-article","created":{"date-parts":[[2018,6,19]],"date-time":"2018-06-19T11:29:34Z","timestamp":1529407774000},"page":"2900-2921","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Differences in substrate specificity of\n                    <i>V. cholerae<\/i>\n                    FabH enzymes suggest new approaches for the development of novel antibiotics and biofuels"],"prefix":"10.1111","volume":"285","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5520-9237","authenticated-orcid":false,"given":"Jing","family":"Hou","sequence":"first","affiliation":[{"name":"Department of Molecular Physiology and Biological Physics University of Virginia Charlottesville VA USA"},{"name":"Center for Structural Genomics of Infectious Diseases (CSGID) Consortium Charlottesville VA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6961-4938","authenticated-orcid":false,"given":"Heping","family":"Zheng","sequence":"additional","affiliation":[{"name":"Department of Molecular Physiology and Biological Physics University of Virginia Charlottesville VA USA"},{"name":"Center for Structural Genomics of Infectious Diseases (CSGID) Consortium Charlottesville VA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wen\u2010Shyong","family":"Tzou","sequence":"additional","affiliation":[{"name":"Department of Molecular Physiology and Biological Physics University of Virginia Charlottesville VA USA"},{"name":"Department of Bioscience and Biotechnology and Center of Excellence for the Oceans National Taiwan Ocean University Keelung Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5240-9789","authenticated-orcid":false,"given":"David R.","family":"Cooper","sequence":"additional","affiliation":[{"name":"Department of Molecular Physiology and Biological Physics University of Virginia Charlottesville VA USA"},{"name":"Center for Structural Genomics of Infectious Diseases (CSGID) Consortium Charlottesville VA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7521-5485","authenticated-orcid":false,"given":"Maksymilian","family":"Chruszcz","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry University of South Carolina Columbia SC USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mahendra D.","family":"Chordia","sequence":"additional","affiliation":[{"name":"Department of Molecular Physiology and Biological Physics University of Virginia Charlottesville VA USA"},{"name":"Center for Structural Genomics of Infectious Diseases (CSGID) Consortium Charlottesville VA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Keehwan","family":"Kwon","sequence":"additional","affiliation":[{"name":"Center for Structural Genomics of Infectious Diseases (CSGID) Consortium Charlottesville VA USA"},{"name":"Infectious Diseases Group J. 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