Structural and biochemical investigation of Metallo-β-lactamases : insights into antibiotic binding sites

Abstract

The emergence of multidrug-resistant (MDR) Gram-negative pathogens and lack of new antibiotics in the pharmaceutical pipeline is seriously limiting our treatment options against common infections. We are now experiencing clinical isolates resistant to virtually all our antibiotics. Our most important and widely used antibiotics, the β-lactams, are challenged by successful β-lactamases. β-lactamases are enzymes produced by bacteria that inactivates the β-lactam antibiotics. The genes for these prosperous β-lactamases are often co-localized with other genes on mobile promiscuous plasmids resulting in MDR isolates of clinically important bacteria such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii. In this thesis we have studied a specific class of β-lactamases termed metallo-β-lactamases (MBLs). MBLs can inactivate virtually all β-lactams including carbapenems which is our most broad-spectrum β-lactams and often reserved for infections that are difficult to treat. So far there is no clinically available inhibitor to MBLs as there is for other β-lactamases. To increase the knowledge about MBLs the three dimensional protein structures of the MBLs, VIM-7, AIM-1, and GIM-1 was determined and the biochemical properties of the MBL TMB-1 were determined. Further, we investigated the interactions between β-lactams and the MBLs using in silico methods. The studies show that although the MBLs share a common three dimensional structure there are differences between the enzymes that contribute to their differences in biochemical activity and substrate specificities. The in silico experiments also show that there are differences in the specific interactions between the various MBLs and different β-lactams. The results from the studies can contribute to the overall understanding of these enzymes and are important for the development of a clinical inhibitor to combat these enzymes which has been successful for other β-lactamases.