Bacterial resistance to antimicrobial agents is a significant threat to the efficacy of antibiotic therapy. The ?-lactam antibiotics, such as the penicillins and cephalosporins, are among the most often used antimicrobials. The most common source of resistance to ?-lactam antibiotics is the production of ?-lactamases, which cleave the drugs and render them ineffective. ?-lactamases have been grouped into four classes based on primary sequence homology. Classes A, C and D are active-site serine enzymes that catalyze the hydrolysis of ?-lactam antibiotics via a serine-bound acyl-enzyme intermediate. Class B enzymes require zinc for activity and catalysis does not proceed via a covalent intermediate. Because of the wide range of substrate specificities of these enzymes, virtually all ?-lactam antibiotics are susceptible to hydrolysis. Clearly, the design of new antibiotics that escape hydrolysis by the growing collection of ?-lactamase activities will be a challenge. It will be necessary to understand the catalytic mechanism and basis for substrate specificity of each class of ?-lactamase. The goal of this work is to understand how the amino acid sequence determines the structure, activity and evolution of class A ?-lactamases. The TEM-1 and CTX-M family of class A enzymes are an important cause of resistance to ?-lactam antibiotics worldwide. In a previous funding period in vitro mutagenesis was used to randomize the entire coding sequence of the class A TEM-1 ?-lactamase. The sets of random mutants were used to identify residues that are critical for the folding, stability and substrate specificity of the enzyme. The recent advent and application of ultra-high throughput sequencing technology has allowed the scope of this approach to be vastly extended to accurately determine the sequence requirements for hydrolysis of multiple classes of ?-lactam antibiotics for every residue position in TEM-1 ?-lactamase. In addition, preliminary studies suggest that amino acid substitutions which increase the stability of TEM-1 ?-lactamase play an important role in the evolution of TEM-1-mediated resistance to extended-spectrum ?-lactam antibiotics and ?-lactamase inhibitors. These studies will be continued to understand how these changes influence the stability and activity of evolved enzyme variants. Finally, the CTX-M family of ?-lactamases exhibits a high degree of sequence variation and it is hypothesized that many of the amino acid substitutions observed alter the stability or substrate specificity of the enzymes. Experiments are proposed to test this hypothesis by examining the role of a selected set of amino acid substitutions on the structure, activity and evolution of CTX-M enzymes.
This project addresses how bacterial resistance to ?-lactam antibiotics such as the penicillins and cephalosporins evolves. ?-lactamases catalyze the destruction of ?-lactam antibiotics and are the most common mechanism of resistance to these drugs. The proposed experiments will determine how amino acid substitutions in the sequence of ?-lactamase enzymes alter their structure, function and evolution, which will facilitate the design of new antimicrobial therapeutics.
|Stojanoski, Vlatko; Adamski, Carolyn J; Hu, Liya et al. (2016) Removal of the Side Chain at the Active-Site Serine by a Glycine Substitution Increases the Stability of a Wide Range of Serine Î²-Lactamases by Relieving Steric Strain. Biochemistry 55:2479-90|
|Stojanoski, Vlatko; Chow, Dar-Chone; Hu, Liya et al. (2015) A triple mutant in the Î©-loop of TEM-1 Î²-lactamase changes the substrate profile via a large conformational change and an altered general base for catalysis. J Biol Chem 290:10382-94|
|Stojanoski, Vlatko; Chow, Dar-Chone; Fryszczyn, Bartlomiej et al. (2015) Structural Basis for Different Substrate Profiles of Two Closely Related Class D Î²-Lactamases and Their Inhibition by Halogens. Biochemistry 54:3370-80|
|Patel, Meha P; Fryszczyn, Bartlomiej G; Palzkill, Timothy (2015) Characterization of the global stabilizing substitution A77V and its role in the evolution of CTX-M Î²-lactamases. Antimicrob Agents Chemother 59:6741-8|
|Mehta, Shrenik C; Rice, Kacie; Palzkill, Timothy (2015) Natural Variants of the KPC-2 Carbapenemase have Evolved Increased Catalytic Efficiency for Ceftazidime Hydrolysis at the Cost of Enzyme Stability. PLoS Pathog 11:e1004949|
|Abriata, Luciano A; Palzkill, Timothy; Dal Peraro, Matteo (2015) How structural and physicochemical determinants shape sequence constraints in a functional enzyme. PLoS One 10:e0118684|
|Adamski, Carolyn J; Cardenas, Ana Maria; Brown, Nicholas G et al. (2015) Molecular basis for the catalytic specificity of the CTX-M extended-spectrum Î²-lactamases. Biochemistry 54:447-57|
|Long, S Wesley; Olsen, Randall J; Mehta, Shrenik C et al. (2014) PBP2a mutations causing high-level Ceftaroline resistance in clinical methicillin-resistant Staphylococcus aureus isolates. Antimicrob Agents Chemother 58:6668-74|
|Fryszczyn, Bartlomiej G; Adamski, Carolyn J; Brown, Nicholas G et al. (2014) Role of Î²-lactamase residues in a common interface for binding the structurally unrelated inhibitory proteins BLIP and BLIP-II. Protein Sci 23:1235-46|
|Dave, Kinjal; Palzkill, Timothy; Pratt, R F (2014) Neutral Î²-Lactams Inactivate High Molecular Mass Penicillin-Binding Proteins of Class B1, Including PBP2a of MRSA. ACS Med Chem Lett 5:154-7|
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