Towards defining mechanisms involved in β-lactam resistance using serial crystallography

Antimicrobial resistance is one of the biggest threats to contemporary medicine, with an increasing number of bacteria showing multi-drug resistance. Bacteria have developed mechanisms to survive β- lactam antibiotic treatment, including by the production of β-lactamases (BLs). BLs hydrolyse the β- lactam ring of vital antibiotics, including penicillins and carbapenems, the latter often described as a “last resort” antibiotic. As described in the Introduction, BLs are divided into two mechanistic groups: serine-β-lactamases, which use a nucleophilic serine to hydrolyse β-lactams (SBLs) and metallo-β- lactamases (MBLs), which use one or two zinc ions to aid hydrolysis. My work focussed on understanding the mechanism by which the MBL Verona Integron metallo-β-lactamase 2 (VIM-2) hydrolyses carbapenems using conventional and serial crystallographic methods. Chapter 2 describes crystallographic, NMR and kinetic studies on VIM-2 and other MBLs and SBLs. Structures of faropenem complexed with VIM-2, the MBL L1 and the SBL KPC-2 (with the University of Bristol) show all three BLs hydrolyse faropenem, with VIM-2 and KPC-2 opening the tetrahydrofuran (THF) side chain. These observations were confirmed by NMR studies, although these also showed that L1 opens the THF ring in solution. Both tautomerisation of the nascent product (enamine to imine) along with epimerisation at the ring junction position were observed. Crystallographic and solution studies with VIM-2 and biapenem indicated the presence of several products, with both imine and enamine complexes being observed at the active site. The work of Chapters 3 and 4 focused on serial crystallographic studies with penicillin binding protein 5 (PBP5) and VIM-2. The aim with VIM-2 was to perform time resolved crystallography using an X-Ray Free Electron Laser (XFEL) to capture the reaction intermediates during carbapenem hydrolysis. PBP5 was initially used to compare and contrast different sample delivery methods for XFEL analyses as it was easier to produce in larger quantities. Multiple resting state datasets of PBP5 and VIM-2 were collected, both at XFELs and at Diamond Light Source using serial data collection; however, no time resolved structures were captured with VIM-2 and the carbapenem ertapenem. A structure of PBP5 in the format of an acyl-enzyme complex with ertapenem was captured, at 2.9 Å resolution. The XFEL studies indicated that VIM-2 is a good candidate for time resolved studies, however, new crystallisation conditions are needed to visualise structural intermediates. Chapter 5 describes structural and solution studies with VIM-2 and three bicyclic boronates, one with a thioether side chain which is a potent, broad spectrum inhibitor of SBLs and MBLs, and two with a sulfonamide type side chain. Overall, the studies performed in this thesis expand our understanding of how VIM-2 hydrolyses carbapenems and penems and of how to potently inhibit a broad range of BLs, including by boronates which are a promising clinically relevant inhibitor class

Studies on the Reactions of Biapenem with VIM Metallo β-Lactamases and the Serine β-Lactamase KPC-2

Carbapenems are important antibacterials and are both substrates and inhibitors of some β-lactamases. We report studies on the reaction of the unusual carbapenem biapenem, with the subclass B1 metallo-β-lactamases VIM-1 and VIM-2 and the class A serine-β-lactamase KPC-2. X-ray diffraction studies with VIM-2 crystals treated with biapenem reveal the opening of the β-lactam ring to form a mixture of the (2S)-imine and enamine complexed at the active site. NMR studies on the reactions of biapenem with VIM-1, VIM-2, and KPC-2 reveal the formation of hydrolysed enamine and (2R)- and (2S)-imine products. The combined results support the proposal that SBL/MBL-mediated carbapenem hydrolysis results in a mixture of tautomerizing enamine and (2R)- and (2S)-imine products, with the thermodynamically favoured (2S)-imine being the major observed species over a relatively long-time scale. The results suggest that prolonging the lifetimes of β-lactamase carbapenem complexes by optimising tautomerisation of the nascently formed enamine to the (2R)-imine and likely more stable (2S)-imine tautomer is of interest in developing improved carbapenems