{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T04:05:52Z","timestamp":1791173152598,"version":"4.1.0"},"reference-count":52,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2008,12,2]],"date-time":"2008-12-02T00:00:00Z","timestamp":1228176000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Protein Science"],"published-print":{"date-parts":[[2009,1]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>The diversity in substrate recognition spectra exhibited by various \u03b2\u2010lactamases can result from one or a few mutations in the active\u2010site area. Using <jats:italic>Escherichia coli<\/jats:italic> TEM\u20101 \u03b2\u2010lactamase as a template that efficiently hydrolyses penicillins, we performed site\u2010saturation mutagenesis simultaneously on two opposite faces of the active\u2010site cavity. Residues 104 and 105 as well as 238, 240, and 244 were targeted to verify their combinatorial effects on substrate specificity and enzyme activity and to probe for cooperativity between these residues. Selection for hydrolysis of an extended\u2010spectrum cephalosporin, cefotaxime (CTX), led to the identification of a variety of novel mutational combinations. <jats:italic>In vivo<\/jats:italic> survival assays and <jats:italic>in vitro<\/jats:italic> characterization demonstrated a general tendency toward increased CTX and decreased penicillin resistance. Although selection was undertaken with CTX, productive binding (<jats:italic>K<\/jats:italic><jats:sub>M<\/jats:sub>) was improved for all substrates tested, including benzylpenicillin for which catalytic turnover (<jats:italic>k<\/jats:italic><jats:sub>cat<\/jats:sub>) was reduced. This indicates broadened substrate specificity, resulting in more generalized (or less specialized) variants. In most variants, the G238S mutation largely accounted for the observed properties, with additional mutations acting in an additive fashion to enhance these properties. However, the most efficient variant did not harbor the mutation G238S but combined two neighboring mutations that acted synergistically, also providing a catalytic generalization. Our exploration of concurrent mutations illustrates the high tolerance of the TEM\u20101 active site to multiple simultaneous mutations and reveals two distinct mutational paths to substrate spectrum diversification.<\/jats:p>","DOI":"10.1002\/pro.25","type":"journal-article","created":{"date-parts":[[2009,2,9]],"date-time":"2009-02-09T23:02:40Z","timestamp":1234220560000},"page":"147-160","source":"Crossref","is-referenced-by-count":24,"title":["High tolerance to simultaneous active\u2010site mutations in TEM\u20101 \u03b2\u2010lactamase: Distinct mutational paths provide more generalized \u03b2\u2010lactam recognition"],"prefix":"10.1002","volume":"18","author":[{"given":"Pierre\u2010Yves","family":"De Wals","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicolas","family":"Doucet","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joelle 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