{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,15]],"date-time":"2026-08-15T08:25:38Z","timestamp":1786782338832,"version":"build-2736575974"},"reference-count":29,"publisher":"Oxford University Press (OUP)","issue":"1","license":[{"start":{"date-parts":[[2022,1,1]],"date-time":"2022-01-01T00:00:00Z","timestamp":1640995200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/journals\/pages\/open_access\/funder_policies\/chorus\/standard_publication_model"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2022,2,24]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:p>CTX-M-15 is a major extended-spectrum beta-lactamase disseminated throughout the globe. The roles of amino acids present in the active-site are widely studied though little is known about the role of the amino acids lying at the close proximity of the CTX-M-15 active-site. Here, by using site-directed mutagenesis we attempted to decipher the role of individual amino acids lying outside the active-site in imparting the beta-lactamase activity of CTX-M-15. Based on the earlier evidence, three amino acid residues namely, Glu169, Asp173 and Arg277 were substituted with alanine. The antibiotic susceptibility of E. coli cells harboring E169A and N173A substituted CTX-M-15 were enhanced by \u223c &amp;gt;32\u00a0fold for penicillins and \u223c 4\u201332 fold for cephalosporins, in comparison to CTX-M-15. However, cells carrying CTX-M-15_R277A did not show a significant difference in antibiotic susceptibility as compared to the wild-type. Further, the catalytic efficiency of the purified CTX-M-15_E169A and CTX-M-15_N173A were compromised when compared with the efficient beta-lactam hydrolysis of purified CTX-M-15. Moreover, the thermal stability of the mutated proteins CTX-M-15_E169A and CTX-M-15_N173A were reduced as compared to the wild type CTX-M-15. Therefore, we conclude that E169 and N173 are crucial non-active-site amino acids that are able to govern the CTX-M-15 activity.<\/jats:p>","DOI":"10.1093\/femsle\/fnac018","type":"journal-article","created":{"date-parts":[[2022,2,15]],"date-time":"2022-02-15T20:24:11Z","timestamp":1644956651000},"source":"Crossref","is-referenced-by-count":6,"title":["Comparative insight into the roles of the non active-site residues E169 and N173 in imparting the beta-lactamase activity of CTX-M-15"],"prefix":"10.1093","volume":"369","author":[{"given":"Jyoti","family":"Verma","sequence":"first","affiliation":[{"name":"Advanced Technology Development Centre, Indian Institute of Technology Kharagpur, Kharagpur-721302, West Bengal, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Diamond","family":"Jain","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur-721302, West Bengal, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dhriti","family":"Mallik","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur-721302, West Bengal, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6403-032X","authenticated-orcid":false,"given":"Anindya S","family":"Ghosh","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur-721302, West Bengal, India"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2022,2,17]]},"reference":[{"key":"2022022502185138600_bib1","doi-asserted-by":"crossref","first-page":"3186","DOI":"10.1016\/S0021-9258(18)49972-3","article-title":"Site-directed mutants, at position 166, of RTEM-1 beta-lactamase that form a stable acyl-enzyme intermediate with penicillin","volume":"266","author":"Adachi","year":"1991","journal-title":"J Biol Chem"},{"key":"2022022502185138600_bib2","doi-asserted-by":"crossref","first-page":"2958","DOI":"10.1128\/AAC.47.9.2958-2961.2003","article-title":"Effects of Ser130gly and Asp240lys substitutions in extended-spectrum \u03b2-lactamase CTX-M-9","volume":"47","author":"Aumeran","year":"2003","journal-title":"Antimicrob Agents Chemother"},{"key":"2022022502185138600_bib3","doi-asserted-by":"crossref","first-page":"1081","DOI":"10.1099\/mic.0.000074","article-title":"A putative low-molecular-mass penicillin-binding protein (PBP) of Mycobacterium smegmatis exhibits prominent physiological characteristics of DD-carboxypeptidase and beta-lactamase","volume":"161","author":"Bansal","year":"2015","journal-title":"Microbiology"},{"key":"2022022502185138600_bib4","doi-asserted-by":"crossref","first-page":"220","DOI":"10.1007\/s10930-017-9713-2","article-title":"A tyrosine residue along with a glutamic acid of the omega-like loop governs the beta-lactamase activity of MSMEG_4455 in Mycobacterium smegmatis","volume":"36","author":"Bansal","year":"2017","journal-title":"Protein J"},{"key":"2022022502185138600_bib5","doi-asserted-by":"crossref","first-page":"2145","DOI":"10.1093\/jac\/dkx146","article-title":"Global epidemiology of CTX-M \u03b2-lactamases: temporal and geographical shifts in genotype","volume":"72","author":"Bevan","year":"2017","journal-title":"J Antimicrob Chemother"},{"key":"2022022502185138600_bib6","doi-asserted-by":"crossref","first-page":"805","DOI":"10.1093\/protein\/4.7.805","article-title":"Site-directed mutagenesis on TEM-1 \u00df-lactamase: role of Glul66 in catalysis and substrate binding","volume":"4","author":"Delaire","year":"1991","journal-title":"Protein Engineering, Design and Selection"},{"key":"2022022502185138600_bib7","doi-asserted-by":"crossref","first-page":"160","DOI":"10.1128\/CMR.00037-09","article-title":"Three decades of \u03b2-lactamase inhibitors","volume":"23","author":"Drawz","year":"2010","journal-title":"Clin Microbiol Rev"},{"key":"2022022502185138600_bib8","doi-asserted-by":"crossref","first-page":"895","DOI":"10.1099\/mic.0.000052","article-title":"A single amino acid substitution in the \u03a9-like loop of e. coli PBP5 disrupts its ability to maintain cell shape and intrinsic beta-lactam resistance","volume":"161","author":"Dutta","year":"2015","journal-title":"Microbiology"},{"key":"2022022502185138600_bib9","doi-asserted-by":"crossref","first-page":"5438","DOI":"10.1074\/jbc.272.9.5438","article-title":"Site-directed mutagenesis of glutamate 166 in two \u03b2-lactamases: kinetic and molecular modeling studies","volume":"272","author":"Guillaume","year":"1997","journal-title":"J Biol Chem"},{"key":"2022022502185138600_bib10","doi-asserted-by":"crossref","first-page":"4121","DOI":"10.1128\/jb.177.14.4121-4130.1995","article-title":"Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter","volume":"177","author":"Guzman","year":"1995","journal-title":"J Bacteriol"},{"key":"2022022502185138600_bib11","doi-asserted-by":"crossref","first-page":"1050","DOI":"10.1093\/jac\/dki130","article-title":"Revised Ambler classification of \u03b2-lactamases","volume":"55","author":"Hall","year":"2005","journal-title":"J Antimicrob Chemother"},{"key":"2022022502185138600_bib12","first-page":"1253","volume-title":"Susceptibility test methods: dilution and disk diffusion methods","author":"Jorgensen","year":"2015","edition":"11th Edition"},{"key":"2022022502185138600_bib13","doi-asserted-by":"crossref","first-page":"9912","DOI":"10.1021\/bi9012098","article-title":"Why the extended-spectrum \u03b2-lactamases SHV-2 and SHV-5 are \u201chypersusceptible\u201d to mechanism-based inhibitors","volume":"48","author":"Kalp","year":"2009","journal-title":"Biochemistry"},{"key":"2022022502185138600_bib14","doi-asserted-by":"crossref","first-page":"fnx259","DOI":"10.1093\/femsle\/fnx259","article-title":"Glutamate residues at positions 162 and 164 influence the beta-lactamase activity of SHV-14 obtained from Klebsiella pneumoniae","volume":"365","author":"Kumar","year":"2018","journal-title":"FEMS Microbiol Lett"},{"key":"2022022502185138600_bib15","doi-asserted-by":"crossref","first-page":"fnx008","DOI":"10.1093\/femsle\/fnx008","article-title":"E152A substitution drastically affects NDM-5 activity","volume":"364","author":"Kumar","year":"2017","journal-title":"FEMS Microbiol Lett"},{"key":"2022022502185138600_bib16","doi-asserted-by":"crossref","first-page":"5325","DOI":"10.1039\/C8RA10313E","article-title":"Significant role of Asn-247 and Arg-64 residues in close proximity of the active site in maintaining the catalytic function of CTX-M-15 type \u03b2-lactamase","volume":"9","author":"Maryam","year":"2019","journal-title":"RSC Adv"},{"key":"2022022502185138600_bib17","doi-asserted-by":"crossref","first-page":"16","DOI":"10.3389\/fmolb.2018.00016","article-title":"Structural and mechanistic basis for extended-spectrum drug-resistance mutations in altering the specificity of TEM, CTX-M, and KPC \u03b2-lactamases","volume":"5","author":"Palzkill","year":"2018","journal-title":"Front Mol Biosci"},{"key":"2022022502185138600_bib18","doi-asserted-by":"crossref","first-page":"657","DOI":"10.1128\/CMR.18.4.657-686.2005","article-title":"Extended-spectrum \u03b2-lactamases: A clinical update","volume":"18","author":"Paterson","year":"2005","journal-title":"Clin Microbiol Rev"},{"key":"2022022502185138600_bib19","doi-asserted-by":"crossref","first-page":"449","DOI":"10.1016\/j.diagmicrobio.2010.11.011","article-title":"Characteristics of infections caused by extended-spectrum \u03b2-lactamase\u2013producing Escherichia coli from community hospitals in South Africa","volume":"69","author":"Peirano","year":"2011","journal-title":"Diagn Microbiol Infect Dis"},{"key":"2022022502185138600_bib20","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1016\/S1473-3099(08)70041-0","article-title":"Extended-spectrum \u03b2-lactamase-producing Enterobacteriaceae: an emerging public-health concern","volume":"8","author":"Pitout","year":"2008","journal-title":"Lancet Infect Dis"},{"key":"2022022502185138600_bib21","doi-asserted-by":"crossref","first-page":"e00891","DOI":"10.1128\/AAC.00891-17","article-title":"Functional characterization of CTX-M-14 and CTX-M-15 \u03b2-lactamases by in vitro DNA shuffling","volume":"61","author":"Po","year":"2017","journal-title":"Antimicrob Agents Chemother"},{"key":"2022022502185138600_bib22","doi-asserted-by":"crossref","first-page":"1031","DOI":"10.1093\/jac\/dkf240","article-title":"Biochemical analysis of the ceftazidime-hydrolysing extended-spectrum \u03b2-lactamase CTX-M-15 and of its structurally related \u03b2-lactamase CTX-M-3","volume":"50","author":"Poirel","year":"2002","journal-title":"J Antimicrob Chemother"},{"key":"2022022502185138600_bib23","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1111\/j.1469-0691.2007.01865.x","article-title":"Genetic support of extended-spectrum \u03b2-lactamases","volume":"14","author":"Poirel","year":"2008","journal-title":"Clin Microbiol Infect"},{"key":"2022022502185138600_bib24","doi-asserted-by":"crossref","first-page":"263","DOI":"10.4103\/0974-777X.68531","article-title":"Extended-spectrum \u03b2-lactamases in Gram Negative Bacteria","volume":"2","author":"Rawat","year":"2010","journal-title":"J Global Infect Dis"},{"key":"2022022502185138600_bib25","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41467-019-08529-y","article-title":"Collateral sensitivity constrains resistance evolution of the CTX-M-15 \u03b2-lactamase","volume":"10","author":"Rosenkilde","year":"2019","journal-title":"Nat Commun"},{"key":"2022022502185138600_bib26","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1111\/j.1469-0691.2007.01867.x","article-title":"The spread of CTX-M-type extended-spectrum \u03b2-lactamases","volume":"14","author":"Rossolini","year":"2008","journal-title":"Clin Microbiol Infect"},{"key":"2022022502185138600_bib27","doi-asserted-by":"crossref","first-page":"6136","DOI":"10.1021\/bi900448u","article-title":"Mutation of the active site carboxy-lysine (K70) of OXA-1 \u03b2-lactamase results in a deacylation-deficient enzyme","volume":"48","author":"Schneider","year":"2009","journal-title":"Biochemistry"},{"key":"2022022502185138600_bib28","doi-asserted-by":"crossref","DOI":"10.3389\/fmicb.2021.688509","article-title":"Structural Comparisons of Cefotaximase (CTX-M-ase) Sub Family 1","volume":"12","author":"Shurina","year":"2021","journal-title":"Front Microbiol"},{"key":"2022022502185138600_bib29","doi-asserted-by":"crossref","first-page":"1124","DOI":"10.1128\/AAC.43.5.1124","article-title":"BOCILLIN FL, a sensitive and commercially available reagent for detection of penicillin-binding proteins","volume":"43","author":"Zhao","year":"1999","journal-title":"Antimicrob Agents Chemother"}],"container-title":["FEMS Microbiology Letters"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/femsle\/advance-article-pdf\/doi\/10.1093\/femsle\/fnac018\/42545927\/fnac018.pdf","content-type":"application\/pdf","content-version":"am","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/femsle\/article-pdf\/369\/1\/fnac018\/42601012\/fnac018.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/femsle\/article-pdf\/369\/1\/fnac018\/42601012\/fnac018.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,2,25]],"date-time":"2022-02-25T02:19:20Z","timestamp":1645755560000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/femsle\/article\/doi\/10.1093\/femsle\/fnac018\/6530193"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022]]},"references-count":29,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2022,2,24]]}},"URL":"https:\/\/doi.org\/10.1093\/femsle\/fnac018","relation":{},"ISSN":["1574-6968"],"issn-type":[{"value":"1574-6968","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2022]]},"published":{"date-parts":[[2022]]},"article-number":"fnac018"}}