{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,15]],"date-time":"2026-05-15T02:40:20Z","timestamp":1778812820856,"version":"3.51.4"},"reference-count":37,"publisher":"Wiley","issue":"10","license":[{"start":{"date-parts":[[2014,8,20]],"date-time":"2014-08-20T00:00:00Z","timestamp":1408492800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"name":"Research Corporation for Science Advancement; Award number:10493;"},{"name":"National Institute of Allergy and Infectious Diseases of the National Institutes of Health","award":["R01 AI100560"],"award-info":[{"award-number":["R01 AI100560"]}]},{"name":"National Institute of Allergy and Infectious Diseases of the National Institutes of Health","award":["R01 AI063517"],"award-info":[{"award-number":["R01 AI063517"]}]},{"name":"Cleveland Department of Veterans Affairs, the Veterans Affairs Merit Review Program","award":["1I01BX001974"],"award-info":[{"award-number":["1I01BX001974"]}]},{"name":"the Geriatric Research Education and Clinical Center VISN 10"},{"name":"W.M. Keck Foundation (support for the Center for Macromolecular Modeling and Materials Design at California State Polytechnic University, Pomona, CA)"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Protein Science"],"published-print":{"date-parts":[[2014,10]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>In Gram\u2010negative bacteria, resistance to \u03b2\u2010lactam antibacterials is largely due to \u03b2\u2010lactamases and is a growing public health threat. One of the most concerning \u03b2\u2010lactamases to evolve in bacteria are the Class B enzymes, the metallo\u2010\u03b2\u2010lactamases (MBLs). To date, penams and cephems resistant to hydrolysis by MBLs have not yet been found. As a result of this broad substrate specificity, a better understanding of the role of catalytically important amino acids in MBLs is necessary to design novel \u03b2\u2010lactams and inhibitors. Two MBLs, the wild type IMP\u20101 with serine at position 262, and an engineered variant with valine at the same position (IMP\u20101\u2010S262V), were previously found to exhibit very different substrate spectra. These findings compelled us to investigate the impact of a threonine at position 262 (IMP\u20101\u2010S262T) on the substrate spectrum. Here, we explore MBL sequence\u2010structure\u2010activity relationships by predicting and experimentally validating the effect of the S262T substitution in IMP\u20101. Using site\u2010directed mutagenesis, threonine was introduced at position 262, and the IMP\u20101\u2010S262T enzyme, as well as the other two enzymes IMP\u20101 and IMP\u20101\u2010S262V, were purified and kinetic constants were determined against a range of \u03b2\u2010lactam antibacterials. Catalytic efficiencies (<jats:italic>k<\/jats:italic><jats:sub>cat<\/jats:sub>\/<jats:italic>K<\/jats:italic><jats:sub>M<\/jats:sub>) obtained with IMP\u20101\u2010S262T and minimum inhibitory concentrations (MICs) observed with bacterial cells expressing the protein were intermediate or comparable to the corresponding values with IMP\u20101 and IMP\u20101\u2010S262V, validating the role of this residue in catalysis. Our results reveal the important role of IMP residue 262 in \u03b2\u2010lactam turnover and support this approach to predict activities of certain novel MBL variants.<\/jats:p>","DOI":"10.1002\/pro.2530","type":"journal-article","created":{"date-parts":[[2014,8,1]],"date-time":"2014-08-01T22:31:32Z","timestamp":1406932292000},"page":"1451-1460","source":"Crossref","is-referenced-by-count":22,"title":["Understanding the determinants of substrate specificity in IMP family metallo\u2010\u03b2\u2010lactamases: The importance of residue 262"],"prefix":"10.1002","volume":"23","author":[{"given":"Kevin M.","family":"Pegg","sequence":"first","affiliation":[{"name":"Biological Sciences Department College of Science, California State Polytechnic University Pomona California"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Eleanor M.","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences College of Pharmacy, Western University of Health Sciences Pomona California"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alex C.","family":"George","sequence":"additional","affiliation":[{"name":"Biological Sciences Department College of Science, California State Polytechnic University Pomona California"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alecander E.","family":"LaCuran","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences College of Pharmacy, Western University of Health Sciences Pomona California"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Christopher R.","family":"Bethel","sequence":"additional","affiliation":[{"name":"Research Service Louis Stokes Cleveland Veterans Affairs Medical Center Cleveland Ohio"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Robert A.","family":"Bonomo","sequence":"additional","affiliation":[{"name":"Research Service Louis Stokes Cleveland Veterans Affairs Medical Center Cleveland Ohio"},{"name":"Departments of Medicine, Pharmacology Biochemistry, and Molecular Biology and Microbiology, Case Western Reserve University Cleveland Ohio"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Peter","family":"Oelschlaeger","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences College of Pharmacy, Western University of Health Sciences Pomona California"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2014,8,20]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1098\/rstb.1980.0049"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.45.3.660-663.2001"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.48.7.2347-2349.2004"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1021\/ar0400241"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bcp.2007.05.021"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1128\/CMR.18.2.306-325.2005"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.2217\/17460913.2.5.501"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.1021\/jm9012938"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/S1473-3099(11)70056-1"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.2174\/138161213804070320"},{"key":"e_1_2_8_12_1","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.38.1.71"},{"key":"e_1_2_8_13_1","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.44.5.1229-1235.2000"},{"key":"e_1_2_8_14_1","doi-asserted-by":"publisher","DOI":"10.1110\/ps.041093405"},{"key":"e_1_2_8_15_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi0300332"},{"key":"e_1_2_8_16_1","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01440-12"},{"key":"e_1_2_8_17_1","doi-asserted-by":"publisher","DOI":"10.1002\/j.1460-2075.1995.tb00174.x"},{"key":"e_1_2_8_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0014-5793(01)02686-2"},{"key":"e_1_2_8_19_1","doi-asserted-by":"publisher","DOI":"10.1016\/0003-2697(85)90409-9"},{"key":"e_1_2_8_20_1","doi-asserted-by":"publisher","DOI":"10.1002\/pro.454"},{"key":"e_1_2_8_21_1","unstructured":"Clinical and Laboratory Standards Institute.Performance standards for antimicrobial susceptibility testing; twenty\u2010second informational supplement. 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