4rx3 | pdb_00004rx3
A triple mutant in the omega-loop of TEM-1 beta-lactamase changes the substrate profile via a large conformational change and an altered general base for catalysis
Structural highlights
FunctionBLAT_ECOLX TEM-type are the most prevalent beta-lactamases in enterobacteria; they hydrolyze the beta-lactam bond in susceptible beta-lactam antibiotics, thus conferring resistance to penicillins and cephalosporins. TEM-3 and TEM-4 are capable of hydrolyzing cefotaxime and ceftazidime. TEM-5 is capable of hydrolyzing ceftazidime. TEM-6 is capable of hydrolyzing ceftazidime and aztreonam. TEM-8/CAZ-2, TEM-16/CAZ-7 and TEM-24/CAZ-6 are markedly active against ceftazidime. IRT-4 shows resistance to beta-lactamase inhibitors. Publication Abstract from PubMedbeta-Lactamases are bacterial enzymes that hydrolyze beta-lactam antibiotics. TEM-1 is a prevalent plasmid-encoded beta-lactamase in Gram-negative bacteria that efficiently catalyzes the hydrolysis of penicillins and early cephalosporins but not oxyimino-cephalosporins. A previous random mutagenesis study identified a W165Y/E166Y/P167G triple mutant that displays greatly altered substrate specificity with increased activity for the oxyimino-cephalosporin, ceftazidime, and decreased activity towards all other beta-lactams tested. Surprisingly, this mutant lacks the conserved Glu166 residue critical for enzyme function. Ceftazidime contains a large, bulky side chain that does not fit optimally in the wild-type TEM-1 active site. Therefore, it was hypothesized that the substitutions in the mutant expand the binding site in the enzyme. To investigate structural changes and address whether there is an enlargement in the active site, the crystal structure of the triple mutant was solved to 1.44-A The structure reveals a large conformational change of the active-site Omega-loop structure to create additional space for the ceftazidime side chain. The position of the hydroxyl group of Tyr166 and an observed shift in the pH profile of the triple mutant suggests that Tyr166 participates in the hydrolytic mechanism of the enzyme. These findings indicate that the highly conserved Glu166 residue can be substituted in the mechanism of serine beta-lactamases. The results reveal the robustness of the overall beta-lactamase fold coupled with the plasticity of an active-site loop facilitate the evolution of enzyme specificity and mechanism. A Triple Mutant in the Omega-loop of TEM-1 beta-lactamase Changes the Substrate Profile via a Large Conformational Change and an Altered General Base for Catalysis.,Stojanoski V, Chow DC, Hu L, Sankaran B, Gilbert HF, Prasad BV, Palzkill T J Biol Chem. 2015 Feb 20. pii: jbc.M114.633438. PMID:25713062[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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