{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T00:47:44Z","timestamp":1789692464058,"version":"4.0.1"},"reference-count":23,"publisher":"Wiley","issue":"9","license":[{"start":{"date-parts":[[2010,7,26]],"date-time":"2010-07-26T00:00:00Z","timestamp":1280102400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"name":"The Veterans Affairs Merit Review Program"},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["RO1 AI063517-01"],"award-info":[{"award-number":["RO1 AI063517-01"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Veterans Integrated Service Network 10 Geriatric Research, Education, and Clinical Center","award":["VISN 10 GRECC"],"award-info":[{"award-number":["VISN 10 GRECC"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Protein Science"],"published-print":{"date-parts":[[2010,9]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>The molecular basis of resistance to \u03b2\u2010lactams and \u03b2\u2010lactam\u2010\u03b2\u2010lactamase inhibitor combinations in the KPC family of class A enzymes is of extreme importance to the future design of effective \u03b2\u2010lactam therapy. Recent crystal structures of KPC\u20102 and other class A \u03b2\u2010lactamases suggest that Ambler position Trp105 may be of importance in binding \u03b2\u2010lactam compounds. Based on this notion, we explored the role of residue Trp105 in KPC\u20102 by conducting site\u2010saturation mutagenesis at this position. <jats:italic>Escherichia coli<\/jats:italic> DH10B cells expressing the Trp105Phe, \u2010Tyr, \u2010Asn, and \u2010His KPC\u20102 variants possessed minimal inhibitory concentrations (MICs) similar to <jats:italic>E<\/jats:italic>. <jats:italic>coli<\/jats:italic> cells expressing wild type (WT) KPC\u20102. Interestingly, most of the variants showed increased MICs to ampicillin\u2010clavulanic acid but not to ampicillin\u2010sulbactam or piperacillin\u2010tazobactam. To explain the biochemical basis of this behavior, four variants (Trp105Phe, \u2010Asn, \u2010Leu, and \u2010Val) were studied in detail. Consistent with the MIC data, the Trp105Phe \u03b2\u2010lactamase displayed improved catalytic efficiencies, <jats:italic>k<\/jats:italic><jats:sub>cat<\/jats:sub>\/<jats:italic>K<\/jats:italic><jats:sub>m<\/jats:sub>, toward piperacillin, cephalothin, and nitrocefin, but slightly decreased <jats:italic>k<\/jats:italic><jats:sub>cat<\/jats:sub>\/<jats:italic>K<\/jats:italic><jats:sub>m<\/jats:sub> toward cefotaxime and imipenem when compared to WT \u03b2\u2010lactamase. The Trp105Asn variant exhibited increased <jats:italic>K<\/jats:italic><jats:sub>m<\/jats:sub>s for all substrates. In contrast, the Trp105Leu and \u2010Val substituted enzymes demonstrated notably decreased catalytic efficiencies (<jats:italic>k<\/jats:italic><jats:sub>cat<\/jats:sub>\/<jats:italic>K<\/jats:italic><jats:sub>m<\/jats:sub>) for all substrates. With respect to clavulanic acid, the <jats:italic>K<\/jats:italic><jats:sub>i<\/jats:sub>s and partition ratios were increased for the Trp105Phe, \u2010Asn, and \u2010Val variants. We conclude that interactions between Trp105 of KPC\u20102 and the \u03b2\u2010lactam are essential for hydrolysis of substrates. Taken together, kinetic and molecular modeling studies define the role of Trp105 in \u03b2\u2010lactam and \u03b2\u2010lactamase inhibitor discrimination.<\/jats:p>","DOI":"10.1002\/pro.454","type":"journal-article","created":{"date-parts":[[2010,7,26]],"date-time":"2010-07-26T16:59:20Z","timestamp":1280163560000},"page":"1714-1727","source":"Crossref","is-referenced-by-count":78,"title":["Elucidating the role of Trp105 in the KPC\u20102 \u03b2\u2010lactamase"],"prefix":"10.1002","volume":"19","author":[{"given":"Krisztina M.","family":"Papp\u2010Wallace","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Magdalena","family":"Taracila","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christopher J.","family":"Wallace","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kristine M.","family":"Hujer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christopher R.","family":"Bethel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John M.","family":"Hornick","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert A.","family":"Bonomo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2010,8,23]]},"reference":[{"key":"e_1_2_7_2_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1469-0691.2008.02101.x"},{"key":"e_1_2_7_3_2","doi-asserted-by":"publisher","DOI":"10.1021\/bi700300u"},{"key":"e_1_2_7_4_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.00961-09"},{"key":"e_1_2_7_5_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.48.6.2321-2324.2004"},{"key":"e_1_2_7_6_2","doi-asserted-by":"publisher","DOI":"10.1128\/CMR.00001-07"},{"key":"e_1_2_7_7_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.45.9.2598-2603.2001"},{"key":"e_1_2_7_8_2","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkn547"},{"key":"e_1_2_7_9_2","doi-asserted-by":"publisher","DOI":"10.1021\/jm9012938"},{"key":"e_1_2_7_10_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.45.4.1151-1161.2001"},{"key":"e_1_2_7_11_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.47.12.3881-3889.2003"},{"key":"e_1_2_7_12_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.00693-09"},{"key":"e_1_2_7_13_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.00163-08"},{"key":"e_1_2_7_14_2","doi-asserted-by":"publisher","DOI":"10.1098\/rstb.1980.0049"},{"key":"e_1_2_7_15_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.00197-10"},{"key":"e_1_2_7_16_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M407606200"},{"key":"e_1_2_7_17_2","doi-asserted-by":"publisher","DOI":"10.1002\/prot.21485"},{"key":"e_1_2_7_18_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M609777200"},{"key":"e_1_2_7_19_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01004-09"},{"key":"e_1_2_7_20_2","doi-asserted-by":"publisher","DOI":"10.1054\/drup.2000.0137"},{"key":"e_1_2_7_21_2","doi-asserted-by":"publisher","DOI":"10.1021\/ja9817996"},{"key":"e_1_2_7_22_2","doi-asserted-by":"publisher","DOI":"10.1021\/ja00134a003"},{"key":"e_1_2_7_23_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M109.011262"},{"key":"e_1_2_7_24_2","volume-title":"CLSI Performance Standards for Antimicrobial Susceptibility Testing. 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