{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T12:26:24Z","timestamp":1790166384586,"version":"4.1.0"},"reference-count":53,"publisher":"American Chemical Society (ACS)","issue":"7","license":[{"start":{"date-parts":[[2019,1,30]],"date-time":"2019-01-30T00:00:00Z","timestamp":1548806400000},"content-version":"vor","delay-in-days":19,"URL":"https:\/\/pubs.acs.org\/page\/policy\/authorchoice_ccbyncnd_termsofuse.html"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2019,2,19]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis (TB), is naturally resistant to \u03b2-lactam antibiotics due to the production of the extended spectrum \u03b2-lactamase BlaC. \u03b2-Lactam\/\u03b2-lactamase inhibitor combination therapies can circumvent the BlaC-mediated resistance of Mtb and are promising treatment options against TB. However, still little is known of the exact mechanism of BlaC inhibition by the \u03b2-lactamase inhibitors currently approved for clinical use, clavulanic acid, sulbactam, tazobactam, and avibactam. Here, we present the X-ray diffraction crystal structures of the acyl-enzyme adducts of wild-type BlaC with the four inhibitors. The +70 Da adduct derived from clavulanate and the trans-enamine acylation adducts of sulbactam and tazobactam are reported. BlaC in complex with avibactam revealed two inhibitor conformations. Preacylation binding could not be observed because inhibitor binding was not detected in BlaC variants carrying a substitution of the active site serine 70 to either alanine or cysteine, by crystallography, ITC or NMR. These results suggest that the catalytic serine 70 is necessary not only for enzyme acylation but also for increasing BlaC affinity for inhibitors in the preacylation state. The structure of BlaC with the serine to cysteine mutation showed a covalent linkage of the cysteine 70 S\u03b3 atom to the nearby amino group of lysine 73. The differences of adduct conformations between BlaC and other \u03b2-lactamases are discussed.<\/jats:p>","DOI":"10.1021\/acs.biochem.8b01085","type":"journal-article","created":{"date-parts":[[2019,1,11]],"date-time":"2019-01-11T09:48:32Z","timestamp":1547200112000},"page":"997-1009","source":"Crossref","is-referenced-by-count":32,"title":["New Conformations of Acylation Adducts of Inhibitors\nof \u03b2-Lactamase from\n                    <i>Mycobacterium tuberculosis<\/i>"],"prefix":"10.1021","volume":"58","author":[{"given":"Raffaella","family":"Tassoni","sequence":"first","affiliation":[{"name":"Leiden University , , Einsteinweg 55 , ,","place":["Leiden, The Netherlands, 2333CC"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anneloes","family":"Blok","sequence":"additional","affiliation":[{"name":"Leiden University , , Einsteinweg 55 , ,","place":["Leiden, The Netherlands, 2333CC"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Navraj S.","family":"Pannu","sequence":"additional","affiliation":[{"name":"Leiden University , , Einsteinweg 55 , ,","place":["Leiden, The Netherlands, 2333CC"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2615-6914","authenticated-orcid":true,"given":"Marcellus","family":"Ubbink","sequence":"additional","affiliation":[{"name":"Leiden University , , Einsteinweg 55 , ,","place":["Leiden, The Netherlands, 2333CC"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2019,1,11]]},"reference":[{"key":"2026090213365382700_cit1","doi-asserted-by":"publisher","first-page":"160","DOI":"10.1128\/CMR.00037-09","article-title":"Three decades of\nb-lactamase inhibitors","volume":"23","author":"Drawz","year":"2010","journal-title":"Clin. Microbiol. Rev."},{"key":"2026090213365382700_cit2","doi-asserted-by":"publisher","first-page":"11998","DOI":"10.1021\/bi701506h","article-title":"Irreversible\ninhibition of the Mycobacterium tuberculosis beta-lactamase by clavulanate","volume":"46","author":"Hugonnet","year":"2007","journal-title":"Biochemistry"},{"key":"2026090213365382700_cit3","doi-asserted-by":"publisher","first-page":"5312","DOI":"10.1021\/bi8001055","article-title":"Structure of the covalent adduct\nformed between Mycobacterium tuberculosis beta-lactamase\nand clavulanate","volume":"47","author":"Tremblay","year":"2008","journal-title":"Biochemistry"},{"key":"2026090213365382700_cit4","doi-asserted-by":"publisher","first-page":"2379","DOI":"10.1093\/jac\/dks231","article-title":"Novel fragments\nof clavulanate observed in the structure of the class A beta-lactamase\nfrom Bacillus licheniformis BS3","volume":"67","author":"Power","year":"2012","journal-title":"J. Antimicrob. Chemother."},{"key":"2026090213365382700_cit5","unstructured":"WHO.\n          . (2018)  Global Tuberculosis Report 2018, ISBN 9789241565646."},{"key":"2026090213365382700_cit6","doi-asserted-by":"publisher","first-page":"668","DOI":"10.7164\/antibiotics.29.668","article-title":"Naturally-occurring beta-lactamase\ninhibitors with antibacterial activity","volume":"29","author":"Brown","year":"1976","journal-title":"J. Antibiot."},{"key":"2026090213365382700_cit7","doi-asserted-by":"publisher","first-page":"852","DOI":"10.1128\/AAC.11.5.852","article-title":"Clavulanic acid: a\nbeta-lactamase-inhibiting beta-lactam from Streptomyces clavuligerus","volume":"11","author":"Reading","year":"1977","journal-title":"Antimicrob.\nAgents Chemother."},{"key":"2026090213365382700_cit8","doi-asserted-by":"publisher","first-page":"2620","DOI":"10.1128\/AAC.39.12.2620","article-title":"Can penicillins and other beta-lactam\nantibiotics be used to treat tuberculosis","volume":"39","author":"Chambers","year":"1995","journal-title":"Antimicrob.\nAgents Chemother."},{"key":"2026090213365382700_cit9","doi-asserted-by":"publisher","first-page":"3384","DOI":"10.1128\/AAC.05690-11","article-title":"Meropenem-clavulanic\nacid shows activity against Mycobacterium tuberculosis in vivo","volume":"56","author":"England","year":"2012","journal-title":"Antimicrob. Agents Chemother."},{"key":"2026090213365382700_cit10","doi-asserted-by":"publisher","first-page":"393","DOI":"10.1128\/AAC.01035-15","article-title":"In vitro activity of\nbeta-lactams in combination with beta-lactamase inhibitors against\nmultidrug-resistant Mycobacterium tuberculosis isolates","volume":"60","author":"Zhang","year":"2016","journal-title":"Antimicrob. Agents Chemother."},{"key":"2026090213365382700_cit11","doi-asserted-by":"publisher","first-page":"393","DOI":"10.1056\/NEJMc1513236","article-title":"\u03b2-Lactams against tuberculosis\n\u2500 New trick for an old dog?","volume":"375","author":"Diacon","year":"2016","journal-title":"N. Engl.\nJ. Med."},{"key":"2026090213365382700_cit12","doi-asserted-by":"publisher","first-page":"1215","DOI":"10.1126\/science.1167498","article-title":"Meropenem-clavulanate\nis effective against extensively drug-resistant Mycobacterium tuberculosis","volume":"323","author":"Hugonnet","year":"2009","journal-title":"Science"},{"key":"2026090213365382700_cit13","doi-asserted-by":"publisher","first-page":"1308","DOI":"10.1128\/AAC.03461-14","article-title":"Rapid cytolysis\nof Mycobacterium tuberculosis by faropenem, an orally\nbioavailable \u03b2-lactam antibiotic","volume":"59","author":"Dhar","year":"2015","journal-title":"Antimicrob.\nAgents Chemother."},{"key":"2026090213365382700_cit14","doi-asserted-by":"publisher","first-page":"6257","DOI":"10.1021\/acs.biochem.7b00556","article-title":"Phosphate promotes the recovery of Mycobacterium tuberculosis beta-lactamase from clavulanic acid inhibition","volume":"56","author":"Elings","year":"2017","journal-title":"Biochemistry"},{"key":"2026090213365382700_cit15","doi-asserted-by":"publisher","first-page":"4551","DOI":"10.1021\/bi300508r","article-title":"NXL104 irreversibly inhibits the\nbeta-lactamase from Mycobacterium tuberculosis","volume":"51","author":"Xu","year":"2012","journal-title":"Biochemistry"},{"key":"2026090213365382700_cit16","doi-asserted-by":"publisher","first-page":"5714","DOI":"10.1128\/AAC.00598-15","article-title":"Hydrolysis of clavulanate\nby Mycobacterium tuberculosis beta-lactamase BlaC\nharboring a canonical SDN motif","volume":"59","author":"Soroka","year":"2015","journal-title":"Antimicrob.\nAgents Chemother."},{"key":"2026090213365382700_cit17","doi-asserted-by":"publisher","first-page":"1103","DOI":"10.1016\/0022-2836(92)90472-V","article-title":"Inhibition of beta-lactamase by clavulanate - trapped\nintermediates in cryocrystallographic studies","volume":"224","author":"Chen","year":"1992","journal-title":"J. Mol. Biol."},{"key":"2026090213365382700_cit18","doi-asserted-by":"publisher","first-page":"34900","DOI":"10.1074\/jbc.M505333200","article-title":"High resolution\ncrystal structures\nof the trans-enamine intermediates formed by sulbactam and clavulanic\nacid and E166A SHV-1 beta-lactamase","volume":"280","author":"Padayatti","year":"2005","journal-title":"J. Biol.\nChem."},{"key":"2026090213365382700_cit19","doi-asserted-by":"publisher","first-page":"2762","DOI":"10.1128\/AAC.00320-06","article-title":"Crystal structure and activity studies\nof the Mycobacterium tuberculosis beta-lactamase\nreveal its critical role in resistance to beta-lactam antibiotics","volume":"50","author":"Wang","year":"2006","journal-title":"Antimicrob. Agents Chemother."},{"key":"2026090213365382700_cit20","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0073123","article-title":"Directed\nevolution of Mycobacterium tuberculosis beta-lactamase\nreveals gatekeeper residue that regulates antibiotic resistance and\ncatalytic efficiency","volume":"8","author":"Feiler","year":"2013","journal-title":"PLoS One"},{"key":"2026090213365382700_cit21","doi-asserted-by":"publisher","first-page":"521","DOI":"10.1099\/mic.0.27629-0","article-title":"Genetic\nanalysis of the beta-lactamases\nof Mycobacterium tuberculosis and Mycobacterium smegmatis and susceptibility to beta-lactam antibiotics","volume":"151","author":"Flores","year":"2005","journal-title":"Microbiology (London, U. K.)"},{"key":"2026090213365382700_cit22","doi-asserted-by":"publisher","first-page":"5657","DOI":"10.1021\/acs.biochem.5b00698","article-title":"Kinetic\nand structural characterization of the interaction\nof 6-methylidene penem 2 with the beta-lactamase from Mycobacterium tuberculosis","volume":"54","author":"Hazra","year":"2015","journal-title":"Biochemistry"},{"key":"2026090213365382700_cit23","doi-asserted-by":"publisher","first-page":"1215","DOI":"10.1126\/science.1167498","article-title":"Meropenem-clavulanate\nis effective against extensively drug-resistant Mycobacterium tuberculosis","volume":"323","author":"Hugonnet","year":"2009","journal-title":"Science"},{"key":"2026090213365382700_cit24","doi-asserted-by":"publisher","first-page":"3766","DOI":"10.1021\/bi100232q","article-title":"Biochemical\nand structural characterization of Mycobacterium tuberculosis beta-Lactamase with the carbapenems\nertapenem and doripenem","volume":"49","author":"Tremblay","year":"2010","journal-title":"Biochemistry"},{"key":"2026090213365382700_cit25","doi-asserted-by":"publisher","first-page":"9685","DOI":"10.1021\/bi1015088","article-title":"Structures\nof the Michaelis complex (1.2 \u00c5) and\nthe covalent acyl intermediate (2.0 \u00c5) of cefamandole bound in\nthe active sites of the Mycobacterium tuberculosis beta-lactamase K73A and E166A mutants","volume":"49","author":"Tremblay","year":"2010","journal-title":"Biochemistry"},{"key":"2026090213365382700_cit26","doi-asserted-by":"publisher","first-page":"234","DOI":"10.1021\/acsinfecdis.5b00003","article-title":"Inhibiting the beta-lactamase of Mycobacterium tuberculosis (Mtb) with novel boronic acid transition-state\ninhibitors (BATSIs)","volume":"1","author":"Kurz","year":"2015","journal-title":"ACS Infect. Dis."},{"key":"2026090213365382700_cit27","doi-asserted-by":"publisher","first-page":"1081","DOI":"10.1093\/jac\/dkw546","article-title":"Inhibition of beta-lactamases\nof mycobacteria by avibactam and clavulanate","volume":"72","author":"Soroka","year":"2016","journal-title":"J. Antimicrob. Chemother."},{"key":"2026090213365382700_cit28","doi-asserted-by":"publisher","first-page":"133","DOI":"10.1107\/S0907444909047374","article-title":"Integration,\nscaling, space-group assignment and post-refinement","volume":"66","author":"Kabsch","year":"2010","journal-title":"Acta Crystallogr., Sect. D: Biol. Crystallogr."},{"key":"2026090213365382700_cit29","doi-asserted-by":"publisher","first-page":"1204","DOI":"10.1107\/S0907444913000061","article-title":"How good\nare my data and what is the resolution?","volume":"69","author":"Evans","year":"2013","journal-title":"Acta\nCrystallogr., Sect. D: Biol. Crystallogr."},{"key":"2026090213365382700_cit30","doi-asserted-by":"publisher","first-page":"1022","DOI":"10.1107\/S0021889897006766","article-title":"MOLREP: an\nautomated program for molecular replacement","volume":"30","author":"Vagin","year":"1997","journal-title":"J. Appl. Crystallogr."},{"key":"2026090213365382700_cit31","doi-asserted-by":"publisher","first-page":"235","DOI":"10.1107\/S0907444910045749","article-title":"Overview\nof the CCP4 suite and current developments","volume":"67","author":"Winn","year":"2011","journal-title":"Acta Crystallogr., Sect. D: Biol. Crystallogr."},{"key":"2026090213365382700_cit32","doi-asserted-by":"publisher","first-page":"355","DOI":"10.1107\/S0907444911001314","article-title":"REFMAC5\nfor the refinement of macromolecular crystal structures","volume":"67","author":"Murshudov","year":"2011","journal-title":"Acta Crystallogr., Sect. D: Biol. Crystallogr."},{"key":"2026090213365382700_cit33","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":"2026090213365382700_cit34","doi-asserted-by":"publisher","first-page":"11895","DOI":"10.1021\/bi060990m","article-title":"Effect of\nthe inhibitor-resistant\nM69V substitution on the structures and populations of trans-enamine\nbeta-lactamase intermediates","volume":"45","author":"Totir","year":"2006","journal-title":"Biochemistry"},{"key":"2026090213365382700_cit35","doi-asserted-by":"publisher","first-page":"1861","DOI":"10.1021\/bi0022745","article-title":"Inhibition of the\nSHV-1 beta-lactamase by sulfones:\nCrystallographic observation of two reaction intermediates with tazobactam","volume":"40","author":"Kuzin","year":"2001","journal-title":"Biochemistry"},{"key":"2026090213365382700_cit36","doi-asserted-by":"publisher","first-page":"843","DOI":"10.1021\/bi035985m","article-title":"Tazobactam\nforms a stoichiometric trans-enamine intermediate in the E166A variant\nof SHV-1 beta-lactamase: 1.63 angstrom crystal structure","volume":"43","author":"Padayatti","year":"2004","journal-title":"Biochemistry"},{"key":"2026090213365382700_cit37","doi-asserted-by":"publisher","first-page":"14111","DOI":"10.1021\/bi0487903","article-title":"Inhibitor-resistant\nclass A beta-lactamases: Consequences of the Ser130-to-Gly mutation\nseen in apo and tazobactam structures of the SHV-1 variant","volume":"43","author":"Sun","year":"2004","journal-title":"Biochemistry"},{"key":"2026090213365382700_cit38","doi-asserted-by":"publisher","first-page":"1437","DOI":"10.1002\/cmdc.201800213","article-title":"Atomic-resolution structure of a\nClass C -lactamase and its complex with avibactam","volume":"13","author":"Pozzi","year":"2018","journal-title":"ChemMedChem"},{"key":"2026090213365382700_cit39","doi-asserted-by":"publisher","first-page":"2496","DOI":"10.1128\/AAC.02247-12","article-title":"Structural insight\ninto potent broad-spectrum inhibition with reversible recyclization\nmechanism: Avibactam in complex with CTX-M-15 and Pseudomonas aeruginosa AmpC beta-lactamases","volume":"57","author":"Lahiri","year":"2013","journal-title":"Antimicrob.\nAgents Chemother."},{"key":"2026090213365382700_cit40","doi-asserted-by":"publisher","first-page":"2252","DOI":"10.1107\/S1600576716014965","article-title":"ContaMiner and ContaBase:\na webserver and database for early identification of unwantedly crystallized\nprotein contaminants","volume":"49","author":"Hungler","year":"2016","journal-title":"J. Appl. Crystallogr."},{"key":"2026090213365382700_cit41","doi-asserted-by":"publisher","first-page":"6114","DOI":"10.1021\/bi300509j","article-title":"Shift in the\nequilibrium between\non and off states of the allosteric switch in Ras-GppNHp affected\nby small molecules and bulk solvent composition","volume":"51","author":"Holzapfel","year":"2012","journal-title":"Biochemistry"},{"key":"2026090213365382700_cit42","doi-asserted-by":"publisher","first-page":"3212","DOI":"10.1107\/S1399004714023827","article-title":"The crystal structure\nof human mitochondrial 3-ketoacyl-CoA\nthiolase (T1): insight into the reaction mechanism of its thiolase\nand thioesterase activities","volume":"70","author":"Kiema","year":"2014","journal-title":"Acta Crystallogr.,\nSect. D: Biol. Crystallogr."},{"key":"2026090213365382700_cit43","doi-asserted-by":"publisher","first-page":"391","DOI":"10.1021\/acs.biochem.6b00906","article-title":"Short carboxylic\nacid-carboxylate\nhydrogen bonds can have fully localized protons","volume":"56","author":"Lin","year":"2017","journal-title":"Biochemistry"},{"key":"2026090213365382700_cit44","doi-asserted-by":"publisher","first-page":"221","DOI":"10.1021\/jm901059x","article-title":"Structure-based\ndesign of pteridine reductase inhibitors targeting African sleeping\nsickness and the Leishmaniases","volume":"53","author":"Tulloch","year":"2010","journal-title":"J. Med. Chem."},{"key":"2026090213365382700_cit45","doi-asserted-by":"publisher","first-page":"747","DOI":"10.1089\/ars.2015.6346","article-title":"Does the transcription\nfactor NemR use a regulatory sulfenamide bond to sense bleach?","volume":"23","author":"Gray","year":"2015","journal-title":"Antioxid. Redox Signaling"},{"key":"2026090213365382700_cit46","doi-asserted-by":"publisher","first-page":"16798","DOI":"10.1021\/ja3073676","article-title":"Crystal structure\nof a preacylation complex of the\nbeta-lactamase inhibitor sulbactam bound to a sulfenamide bond-containing\nthiol-beta-lactamase","volume":"134","author":"Rodkey","year":"2012","journal-title":"J. Am. Chem. Soc."},{"key":"2026090213365382700_cit47","doi-asserted-by":"publisher","first-page":"709","DOI":"10.1021\/bi061546m","article-title":"Reversible oxidation\nof the membrane distal domain of receptor PTP alpha is mediated by\na cyclic sulfenamide","volume":"46","author":"Yang","year":"2007","journal-title":"Biochemistry"},{"key":"2026090213365382700_cit48","doi-asserted-by":"publisher","first-page":"395","DOI":"10.1111\/mmi.12192","article-title":"Novel regulatory\nsystem nemRA-gloA\nfor electrophile reduction in Escherichia coli K-12","volume":"88","author":"Lee","year":"2013","journal-title":"Mol. Microbiol."},{"key":"2026090213365382700_cit49","first-page":"141","article-title":"Bacterial responses to reactive chlorine species","volume-title":"Annu. Rev. Microbiol.","author":"Gray","year":"2013"},{"key":"2026090213365382700_cit50","doi-asserted-by":"publisher","first-page":"12421","DOI":"10.1021\/bi961044g","article-title":"Inhibition of TEM-2 beta-lactamase\nfrom Escherichia coli by clavulanic acid: Observation\nof intermediates by electrospray ionization mass spectrometry","volume":"35","author":"Brown","year":"1996","journal-title":"Biochemistry"},{"key":"2026090213365382700_cit51","doi-asserted-by":"publisher","first-page":"32149","DOI":"10.1074\/jbc.M204212200","article-title":"The structural bases of antibiotic\nresistance in the clinically derived mutant beta-lactamases TEM-30,\nTEM-32, and TEM-34","volume":"277","author":"Wang","year":"2002","journal-title":"J. Biol. Chem."},{"key":"2026090213365382700_cit52","doi-asserted-by":"publisher","first-page":"52724","DOI":"10.1074\/jbc.M306059200","article-title":"Understanding resistance\nto beta-lactams and beta-lactamase\ninhibitors in the SHV beta-lactamase - Lessons from the mutagenesis\nof Ser-130","volume":"278","author":"Helfand","year":"2003","journal-title":"J. Biol. Chem."},{"key":"2026090213365382700_cit53","doi-asserted-by":"publisher","first-page":"87","DOI":"10.1111\/febs.14315","article-title":"Mechanisms\nof proton relay and product release by Class A beta-lactamase\nat ultrahigh resolution","volume":"285","author":"Lewandowski","year":"2018","journal-title":"FEBS J."}],"container-title":["Biochemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.biochem.8b01085","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/bichaw\/article-pdf\/58\/7\/997\/66943687\/acs.biochem.8b01085.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/bichaw\/article-pdf\/58\/7\/997\/66943687\/acs.biochem.8b01085.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T17:37:00Z","timestamp":1788370620000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/bichaw\/article\/58\/7\/997\/587533\/New-Conformations-of-Acylation-Adducts-of"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,1,11]]},"references-count":53,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2019,1,11]]},"published-print":{"date-parts":[[2019,2,19]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.biochem.8b01085","relation":{},"ISSN":["0006-2960","1520-4995"],"issn-type":[{"value":"0006-2960","type":"print"},{"value":"1520-4995","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,1,11]]}}}