The β-lactams retain a central place in the antibacterial armamentarium. In Gram-negative bacteria, β-lactamase enzymes that hydrolyze the amide bond of the four-membered β-lactam ring are the primary resistance mechanism, with multiple enzymes disseminating on mobile genetic elements across opportunistic pathogens such as Enterobacteriaceae (e.g., Escherichia coli ) and non-fermenting organisms (e.g., Pseudomonas aeruginosa ). β-Lactamases divide into four classes; the active-site serine β-lactamases (classes A, C and D) and the zinc-dependent or metallo-β-lactamases (MBLs; class B). Here we review recent advances in mechanistic understanding of each class, focusing upon how growing numbers of crystal structures, in particular for β-lactam complexes, and methods such as neutron diffraction and molecular simulations, have improved understanding of the biochemistry of β-lactam breakdown. A second focus is β-lactamase interactions with carbapenems, as carbapenem-resistant bacteria are of grave clinical concern and carbapenem-hydrolyzing enzymes such as KPC (class A) NDM (class B) and OXA-48 (class D) are proliferating worldwide. An overview is provided of the changing landscape of β-lactamase inhibitors, exemplified by the introduction to the clinic of combinations of β-lactams with diazabicyclooctanone and cyclic boronate serine β-lactamase inhibitors, and of progress and strategies toward clinically useful MBL inhibitors. Despite the long history of β-lactamase research, we contend that issues including continuing unresolved questions around mechanism; opportunities afforded by new technologies such as serial femtosecond crystallography; the need for new inhibitors, particularly for MBLs; the likely impact of new β-lactam:inhibitor combinations and the continuing clinical importance of β-lactams mean that this remains a rewarding research area.

The polymixin colistin is a “last line” antibiotic against extensively-resistant Gram-negative bacteria. Recently, the mcr-1 gene was identified as a plasmid-mediated resistance mechanism in human and animal Enterobacteriaceae, with a wide geographical distribution and many producer strains resistant to multiple other antibiotics. mcr-1 encodes a membrane-bound enzyme catalysing phosphoethanolamine transfer onto bacterial lipid A. Here we present crystal structures revealing the MCR-1 periplasmic, catalytic domain to be a zinc metalloprotein with an alkaline phosphatase/sulphatase fold containing three disulphide bonds. One structure captures a phosphorylated form representing the first intermediate in the transfer reaction. Mutation of residues implicated in zinc or phosphoethanolamine binding, or catalytic activity, restores colistin susceptibility of recombinant E. coli. Zinc deprivation reduces colistin MICs in MCR-1-producing laboratory, environmental, animal and human E. coli. Conversely, over-expression of the disulphide isomerase DsbA increases the colistin MIC of laboratory E. coli. Preliminary density functional theory calculations on cluster models suggest a single zinc ion may be sufficient to support phosphoethanolamine transfer. These data demonstrate the importance of zinc and disulphide bonds to MCR-1 activity, suggest that assays under zinc-limiting conditions represent a route to phenotypic identification of MCR-1 producing E. coli, and identify key features of the likely catalytic mechanism.

Metallo-β-lactamases (MBLs) hydrolyze almost all β-lactam antibiotics and are unaffected by clinically available β-lactamase inhibitors (βLIs). Active-site architecture divides MBLs into three classes (B1, B2, and B3), complicating development of βLIs effective against all enzymes. Bisthiazolidines (BTZs) are carboxylate-containing, bicyclic compounds, considered as penicillin analogs with an additional free thiol. Here, we show both L-and D-BTZ enantiomers are micromolar competitive βLIs of all MBL classes in vitro, with K i s of 6-15 μM or 36-84 μM for subclass B1 MBLs (IMP-1 and BcII, respectively), and 10-12 μM for the B3 enzyme L1. Against the B2 MBL Sfh-I, the L-BTZ enantiomers exhibit 100-fold lower K i s (0.26-0.36 μM) than D-BTZs (26-29 μM). Importantly, cell-based time-kill assays show BTZs restore β-lactam susceptibility of Escherichia coli-producing MBLs (IMP-1, Sfh-1, BcII, and GOB-18) and, significantly, an extensively drug-resistant Stenotrophomonas maltophilia clinical isolate expressing L1. BTZs therefore inhibit the full range of MBLs and potentiate β-lactam activity against producer pathogens. X-ray crystal structures reveal insights into diverse BTZ binding modes, varying with orientation of the carboxylate and thiol moieties. BTZs bind the di-zinc centers of B1 (IMP-1; BcII) and B3 (L1) MBLs via the free thiol, but orient differently depending upon stereochemistry. In contrast, the L-BTZ carboxylate dominates interactions with the monozinc B2 MBL Sfh-I, with the thiol uninvolved. D-BTZ complexes most closely resemble β-lactam binding to B1 MBLs, but feature an unprecedented disruption of the D120-zinc interaction. Cross-class MBL inhibition therefore arises from the unexpected versatility of BTZ binding.carbapenemase | antibiotic resistance | inhibitors | bisthiazolidines | metallo-β-lactamase

Class A serine β-lactamases (SBLs) are key antibiotic resistance determinants in Gram-negative bacteria. SBLs neutralize β-lactams via a hydrolytically labile covalent acyl-enzyme intermediate. Klebsiella pneumoniae carbapenemase (KPC) is a widespread SBL that hydrolyzes carbapenems, the most potent β-lactams; known KPC variants differ in turnover of expanded-spectrum oxyimino-cephalosporins (ESOCs), e.g. cefotaxime and ceftazidime. Here, we compare ESOC hydrolysis by the parent enzyme KPC-2 and its clinically observed double variant (P104R/V240G) KPC-4. Kinetic analyses show KPC-2 hydrolyzes cefotaxime more efficiently than the bulkier ceftazidime, with improved ESOC turnover by KPC-4 resulting from enhanced turnover (kcat), rather than binding (KM). High-resolution crystal structures of ESOC acyl-enzyme complexes with deacylation-deficient (E166Q) KPC-2 and KPC-4 mutants show that ceftazidime acylation causes rearrangement of three loops; the Ω-, 240- and 270-loops, that border the active site. However, these rearrangements are less pronounced in the KPC-4 than the KPC-2 ceftazidime acyl-enzyme, and are not observed in the KPC-2:cefotaxime acyl-enzyme. Molecular dynamics simulations of KPC:ceftazidime acyl-enyzmes reveal that the deacylation general base E166, located on the Ω-loop, adopts two distinct conformations in KPC-2, either pointing ‘in’ or ‘out’ of the active site; with only the ‘in’ form compatible with deacylation. The ‘out’ conformation was not sampled in the KPC-4 acyl-enzyme, indicating that efficient ESOC breakdown is dependent upon the ordering and conformation of the KPC Ω-loop. The results explain how point mutations expand the activity spectrum of the clinically important KPC SBLs to include ESOCs through their effects on the conformational dynamics of the acyl-enzyme intermediate.

Understanding allostery in enzymes and tools to identify it, offer promising alternative strategies to inhibitor development. Through a combination of equilibrium and nonequilibrium molecular dynamics simulations, we identify allosteric effects and communication pathways in two prototypical class A β-lactamases, TEM-1 and KPC-2, which are important determinants of antibiotic resistance. The nonequilibrium simulations reveal pathways of communication operating over distances of 30 Å or more. Propagation of the signal occurs through cooperative coupling of loop dynamics. Notably, 50% or more of clinically relevant amino acid substitutions map onto the identified signal transduction pathways. This suggests that clinically important variation may affect, or be driven by, differences in allosteric behavior, providing a mechanism by which amino acid substitutions may affect the relationship between spectrum of activity, catalytic turnover and potential allosteric behavior in this clinically important enzyme family. Simulations of the type presented here will help in identifying and analyzing such differences.