{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T09:28:22Z","timestamp":1787045302024,"version":"build-2736575974"},"reference-count":30,"publisher":"American Chemical Society (ACS)","issue":"28","license":[{"start":{"date-parts":[[2017,6,21]],"date-time":"2017-06-21T00:00:00Z","timestamp":1498003200000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2017,6,21]],"date-time":"2017-06-21T00:00:00Z","timestamp":1498003200000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2017,6,21]],"date-time":"2017-06-21T00:00:00Z","timestamp":1498003200000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["GM115921"],"award-info":[{"award-number":["GM115921"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2017,7,18]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>It has become increasingly apparent within the last several years that unusual N-formylated sugars are often found on the O-antigens of such Gram negative pathogenic organisms as Francisella tularensis, Campylobacter jejuni, and Providencia alcalifaciens, among others. Indeed, in some species of Brucella, for example, the O-antigen contains 1,2-linked 4-formamido-4,6-dideoxy-\u03b1-d-mannosyl groups. These sugars, often referred to as N-formylperosamine, are synthesized in pathways initiating with GDP-mannose. One of the enzymes required for the production of N-formylperosamine, namely, WbkC, was first identified in 2000 and was suggested to function as an N-formyltransferase. Its biochemical activity was never experimentally verified, however. Here we describe a combined structural and functional investigation of WbkC from Brucella melitensis. Four high resolution X-ray structures of WbkC were determined in various complexes to address its active site architecture. Unexpectedly, the quaternary structure of WbkC was shown to be different from that previously observed for other sugar N-formyltransferases. Additionally, the structures revealed a second binding site for a GDP molecule distinct from that required for GDP-perosamine positioning. In keeping with this additional binding site, kinetic data with the wild type enzyme revealed complex patterns. Removal of GDP binding by mutating Phe 142 to an alanine residue resulted in an enzyme variant displaying normal Michaelis\u2013Menten kinetics. These data suggest that this nucleotide binding pocket plays a role in enzyme regulation. Finally, by using an alternative substrate, we demonstrate that WbkC can be utilized to produce a trideoxysugar not found in nature.<\/jats:p>","DOI":"10.1021\/acs.biochem.7b00494","type":"journal-article","created":{"date-parts":[[2017,6,21]],"date-time":"2017-06-21T12:57:14Z","timestamp":1498049834000},"page":"3657-3668","source":"Crossref","is-referenced-by-count":9,"title":["Biochemical Characterization of WbkC, an\n                    <i>N<\/i>\n                    \u2011Formyltransferase from\n                    <i>Brucella melitensis<\/i>"],"prefix":"10.1021","volume":"56","author":[{"given":"Alexander S.","family":"Riegert","sequence":"first","affiliation":[{"name":"University of Wisconsin , , , ,","place":["Madison, Wisconsin, United States, 53706"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel P.","family":"Chantigian","sequence":"additional","affiliation":[{"name":"University of Wisconsin , , , ,","place":["Madison, Wisconsin, United States, 53706"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"James B.","family":"Thoden","sequence":"additional","affiliation":[{"name":"University of Wisconsin , , , ,","place":["Madison, Wisconsin, United States, 53706"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peter A.","family":"Tipton","sequence":"additional","affiliation":[{"name":"University of Missouri , , , ,","place":["Columbia, Missouri, United States, 65211"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6214-3638","authenticated-orcid":true,"given":"Hazel M.","family":"Holden","sequence":"additional","affiliation":[{"name":"University of Wisconsin , , , ,","place":["Madison, Wisconsin, United States, 53706"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2017,6,21]]},"reference":[{"key":"2026081800080421400_cit1","doi-asserted-by":"publisher","first-page":"283","DOI":"10.1093\/clinids\/21.2.283","article-title":"An overview\nof human brucellosis","volume":"21","author":"Young","year":"1995","journal-title":"Clin. Infect. Dis."},{"key":"2026081800080421400_cit2","doi-asserted-by":"publisher","first-page":"392","DOI":"10.1016\/j.vetmic.2009.06.021","article-title":"Brucellosis:\na re-emerging zoonosis","volume":"140","author":"Seleem","year":"2010","journal-title":"Vet. Microbiol."},{"issue":"Suppl 1","key":"2026081800080421400_cit3","doi-asserted-by":"publisher","first-page":"29","DOI":"10.1016\/j.ijantimicag.2007.06.011","article-title":"Challenges\nin Brucella bacteraemia","volume":"30","author":"Pappas","year":"2007","journal-title":"Int.\nJ. Antimicrob. Agents"},{"key":"2026081800080421400_cit4","doi-asserted-by":"publisher","first-page":"209","DOI":"10.1016\/S0923-2508(00)00141-8","article-title":"Conservation of seven genes involved\nin the biosynthesis of the lipopolysaccharide O-side chain in Brucella spp","volume":"151","author":"Cloeckaert","year":"2000","journal-title":"Res. Microbiol."},{"key":"2026081800080421400_cit5","doi-asserted-by":"publisher","first-page":"13","DOI":"10.1186\/1475-2859-5-13","article-title":"Brucella spp noncanonical LPS: structure,\nbiosynthesis, and interaction with host immune system","volume":"5","author":"Cardoso","year":"2006","journal-title":"Microb. Cell Fact."},{"key":"2026081800080421400_cit6","doi-asserted-by":"publisher","first-page":"655","DOI":"10.1016\/S0923-2508(00)90130-X","article-title":"Genetic organisation\nof the lipopolysaccharide O-antigen biosynthesis region of Brucella melitensis 16M (wbk)","volume":"151","author":"Godfroid","year":"2000","journal-title":"Res.\nMicrobiol."},{"key":"2026081800080421400_cit7","doi-asserted-by":"publisher","first-page":"6114","DOI":"10.1021\/bi4009006","article-title":"Structure of a sugar N-formyltransferase from Campylobacter jejuni","volume":"52","author":"Thoden","year":"2013","journal-title":"Biochemistry"},{"key":"2026081800080421400_cit8","doi-asserted-by":"publisher","first-page":"273","DOI":"10.1002\/pro.2409","article-title":"Three-dimensional\nstructure of a sugar N-formyltransferase from Francisella tularensis","volume":"23","author":"Zimmer","year":"2014","journal-title":"Protein Sci."},{"key":"2026081800080421400_cit9","doi-asserted-by":"publisher","first-page":"631","DOI":"10.1021\/bi501539a","article-title":"New role\nfor the ankyrin repeat revealed by a study of the N-formyltransferase from Providencia alcalifaciens","volume":"54","author":"Woodford","year":"2015","journal-title":"Biochemistry"},{"key":"2026081800080421400_cit10","doi-asserted-by":"publisher","first-page":"976","DOI":"10.1002\/pro.2675","article-title":"Molecular structure of an N-formyltransferase\nfrom Providencia alcalifaciens O30","volume":"24","author":"Genthe","year":"2015","journal-title":"Protein Sci."},{"key":"2026081800080421400_cit11","doi-asserted-by":"publisher","first-page":"1555","DOI":"10.1002\/pro.2938","article-title":"Structure of the Escherichia\ncoli ArnA N-formyltransferase domain in\ncomplex with N(5)-formyltetrahydrofolate and UDP-Ara4N","volume":"25","author":"Genthe","year":"2016","journal-title":"Protein Sci."},{"key":"2026081800080421400_cit12","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.sbi.2016.04.003","article-title":"Enzymes required for the biosynthesis\nof N-formylated sugars","volume":"41","author":"Holden","year":"2016","journal-title":"Curr.\nOpin. Struct. Biol."},{"key":"2026081800080421400_cit13","doi-asserted-by":"publisher","first-page":"5627","DOI":"10.1016\/j.vaccine.2010.06.023","article-title":"Inactivation of formyltransferase (wbkC) gene generates\na Brucella abortus rough strain that is attenuated\nin macrophages and in mice","volume":"28","author":"Lacerda","year":"2010","journal-title":"Vaccine"},{"key":"2026081800080421400_cit14","doi-asserted-by":"publisher","first-page":"859","DOI":"10.1107\/S0907444906019949","article-title":"HKL-3000: the integration of data\nreduction and structure solution-from diffraction images to an initial\nmodel in minutes","volume":"62","author":"Minor","year":"2006","journal-title":"Acta Crystallogr., Sect. D:\nBiol. Crystallogr."},{"issue":"4","key":"2026081800080421400_cit15","doi-asserted-by":"publisher","first-page":"849","DOI":"10.1107\/S0907444999000839","article-title":"Automated MAD and MIR structure solution","volume":"55","author":"Terwilliger","year":"1999","journal-title":"Acta Crystallogr., Sect. D: Biol. Crystallogr."},{"issue":"8","key":"2026081800080421400_cit16","doi-asserted-by":"publisher","first-page":"965","DOI":"10.1107\/S0907444900005072","article-title":"Maximum-likelihood density modification","volume":"56","author":"Terwilliger","year":"2000","journal-title":"Acta Crystallogr., Sect. D: Biol. Crystallogr."},{"key":"2026081800080421400_cit17","doi-asserted-by":"publisher","first-page":"2126","DOI":"10.1107\/S0907444904019158","article-title":"Coot: model-building\ntools for molecular graphics","volume":"60","author":"Emsley","year":"2004","journal-title":"Acta Crystallogr.,\nSect. D: Biol. Crystallogr."},{"key":"2026081800080421400_cit18","doi-asserted-by":"publisher","first-page":"240","DOI":"10.1107\/S0907444996012255","article-title":"Refinement of macromolecular\nstructures by the maximum-likelihood method","volume":"53","author":"Murshudov","year":"1997","journal-title":"Acta Crystallogr., Sect. D: Biol. Crystallogr."},{"key":"2026081800080421400_cit19","doi-asserted-by":"publisher","first-page":"658","DOI":"10.1107\/S0021889807021206","article-title":"Phaser\ncrystallographic software","volume":"40","author":"McCoy","year":"2007","journal-title":"J. Appl. Crystallogr."},{"key":"2026081800080421400_cit20","doi-asserted-by":"publisher","first-page":"2833","DOI":"10.1021\/bi702430d","article-title":"GDP-perosamine synthase:\nstructural analysis and production of a novel trideoxysugar","volume":"47","author":"Cook","year":"2008","journal-title":"Biochemistry"},{"key":"2026081800080421400_cit21","doi-asserted-by":"crossref","first-page":"5485","DOI":"10.1128\/IAI.66.11.5485-5493.1998","article-title":"Identification\nof the perosamine synthetase gene of Brucella melitensis 16M and involvement of lipopolysaccharide O side chain in Brucella survival in mice and in macrophages","volume":"66","author":"Godfroid","year":"1998","journal-title":"Infect. Immun."},{"key":"2026081800080421400_cit22","doi-asserted-by":"publisher","first-page":"5328","DOI":"10.1021\/bi047384g","article-title":"Crystal structure and mechanism of\nthe Escherichia coli ArnA (PmrI) transformylase domain.\nAn enzyme for lipid A modification with 4-amino-4-deoxy-l-arabinose and polymyxin resistance","volume":"44","author":"Gatzeva-Topalova","year":"2005","journal-title":"Biochemistry"},{"key":"2026081800080421400_cit23","doi-asserted-by":"crossref","DOI":"10.1007\/978-3-642-80966-8","volume-title":"Initial Rate Enzyme Kinetics","author":"Fromm","year":"1975"},{"key":"2026081800080421400_cit24","doi-asserted-by":"publisher","first-page":"929","DOI":"10.1016\/j.str.2005.03.018","article-title":"Structure and mechanism of ArnA:\nconformational change implies ordered dehydrogenase mechanism in key\nenzyme for polymyxin resistance","volume":"13","author":"Gatzeva-Topalova","year":"2005","journal-title":"Structure"},{"key":"2026081800080421400_cit25","doi-asserted-by":"publisher","first-page":"23000","DOI":"10.1074\/jbc.M501534200","article-title":"Structure and function of both domains\nof ArnA, a dual function decarboxylase and a formyltransferase, involved\nin 4-amino-4-deoxy-l-arabinose biosynthesis","volume":"280","author":"Williams","year":"2005","journal-title":"J. Biol. Chem."},{"key":"2026081800080421400_cit26","doi-asserted-by":"publisher","first-page":"14154","DOI":"10.1074\/jbc.M414265200","article-title":"A formyltransferase\nrequired for polymyxin resistance in Escherichia coli and the modification of lipid A with 4-amino-4-deoxy-l-arabinose.\nIdentification and function of UDP-4-deoxy-4-formamido-l-arabinose","volume":"280","author":"Breazeale","year":"2005","journal-title":"J. Biol. Chem."},{"key":"2026081800080421400_cit27","doi-asserted-by":"publisher","DOI":"10.1016\/j.jsb.2017.03.002","article-title":"Molecular architecture of an N-formyltransferase\nfrom Salmonella enterica O60","author":"Woodford","year":"2017","journal-title":"J. Struct. Biol."},{"key":"2026081800080421400_cit28","doi-asserted-by":"publisher","first-page":"1049","DOI":"10.1006\/jmbi.1993.1351","article-title":"Main-chain\nbond lengths and bond angles in protein structures","volume":"231","author":"Laskowski","year":"1993","journal-title":"J. Mol. Biol."},{"key":"2026081800080421400_cit29","author":"DeLano","year":"2002"},{"key":"2026081800080421400_cit30","doi-asserted-by":"publisher","first-page":"131","DOI":"10.1016\/0022-2836(81)90098-X","article-title":"On the detection of homotropic effects\nin enzymes of low co-operativity. Application to modified aspartate\ntranscarbamoylase","volume":"152","author":"Hensley","year":"1981","journal-title":"J. Mol. Biol."}],"container-title":["Biochemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.biochem.7b00494","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/bichaw\/article-pdf\/56\/28\/3657\/16003083\/bi7b00494.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/bichaw\/article-pdf\/56\/28\/3657\/16003083\/bi7b00494.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T08:43:10Z","timestamp":1787042590000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/bichaw\/article\/56\/28\/3657\/810920\/Biochemical-Characterization-of-WbkC-an-N"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2017,6,21]]},"references-count":30,"journal-issue":{"issue":"28","published-online":{"date-parts":[[2017,6,21]]},"published-print":{"date-parts":[[2017,7,18]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.biochem.7b00494","relation":{},"ISSN":["0006-2960","1520-4995"],"issn-type":[{"value":"0006-2960","type":"print"},{"value":"1520-4995","type":"electronic"}],"subject":[],"published":{"date-parts":[[2017,6,21]]}}}