Bacterial resistance to β-lactam antibiotics is a major public health threat that has prompted the search for inhibitors of β-lactamase enzymes. The development of new inhibitors relies on an understanding of the structural and functional contributions of amino acids in the active site. Previous work has shown that the single mutant Asn152Ser causes class C beta-lactamases (e.g., P99, AmpC, CMY-2) to alter their substrate specificity and susceptibility toward inhibitors. A neighboring residue, Gln120, is seen in X-ray crystal structures to form a hydrogen bond to the C3 carboxylate of cephalosporins and cephamycins, when these antibiotics are bound in a product-like conformation that is thought to precede deacylation. To examine the role of Gln120 in cephalosporin turnover, a site-saturation mutant library was generated in Enterobacter cloacae P99 cephalosporinase to test the effect of all 20 amino acids at this position. Individual library members were screened in vivo for activity against a panel of cephalosporins. P99 variants showing altered substrate specificity were expressed, purified, and kinetically characterized to assess the effect of mutation.

Exploring the role of Gln120 in Enterobacter cloacae P99 cephalosporinase by site-saturation mutagenesis

253rd National Meeting (2017)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.