Mechanistic enzymology of beta-lactam antibiotic resistance mechanisms and target proteins

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Serine beta-lactamases (SBLs) are bacterial enzymes that degrade beta-lactam antibiotics (e.g., penicillins and cephalosporins), contributing to antibiotic resistance. Our research program is dedicated to understanding the catalytic mechanisms of the SBLs, and characterizing how SBLs evolve to degrade new beta-lactams. Most SBLs cannot degrade carbapenems, an important class of beta-lactam antibiotic, and several features of the carbapenem structure are thought to interfere with SBL catalysis. However, in recent years, new SBLs have been identified that efficiently degrade carbapenems. We will investigate how these SBL carbapenemases degrade carbapenems, focusing on interactions involving the carbapenem hydroxyethyl side chain and pyrroline ring. In combination with molecular biology and biochemistry techniques, we will use 19F NMR spectroscopy to study dynamic aspects of the catalytic mechanisms of these enzymes. Based on our recent work, we have identified carbapenem derivatives that are likely to inhibit SBL carbapenemases. We will test these derivatives as SBL inhibitors, using 19F NMR to characterize the mechanistic details of these interactions. In addition to the proposed mechanistic and inhibition studies, we will examine how SBLs might have evolved to develop carbapenemase activity, and test whether putative SBLs encoded in microbiome and environmental samples possess carbapenemase activity. Our research aims to define the mechanisms of SBL carbapenemase catalysis, which will assist the development of new beta-lactam antibiotics that are resistant to SBL-catalyzed degradation. The 19F NMR methods that we develop during the course of this work will enable future mechanistic studies on the degradation of other beta-lactams (e.g., cephalosporins) by SBLs, and can be readily applied to other enzyme-substrate systems. More generally, our proposed research program provides an excellent model for investigating how enzymes evolve to overcome inhibitors, an area of great economic importance in agriculture and veterinary medicine (e.g., evolution of herbicide-resistant weeds, antibiotic-resistant bacteria infecting livestock). Detailed mechanistic studies, such as those proposed herein, are proving to be an essential consideration for future inhibitor development. In addition to benefitting academic and industrial efforts focused on antibiotic and inhibitor development, this research program will benefit the Canadian scientific community by providing 8 highly qualified personnel (1 PhD, 2 MSc, and 5 USRA students) with valuable and sought-after research skills in chemistry, biochemistry and microbiology. This will provide future Canadian scientific leaders with a strong research base from which they can promote future research and development in the natural sciences.