Biology subjects

Tooke, C. L.

Publications and source records attributed to Tooke, C. L..

10 recordsLinked to original sources

bioRxiv2026

Acyl-enzyme dynamics, tautomerisation and hydration regulate turnover of carbapenem antibiotics by the OXA-48 β-lactamase

OXA-48 is a globally disseminated class D serine {beta}-lactamase that efficiently confers resistance to a range of {beta}-lactam antibiotics, including carbapenems, the most potent such agents versus Enterobacterales (Escherichia coli and relatives). Here we characterise the interactions of OXA-48 with the acyl-enzyme complex intermediates formed on its reaction with the carbapenems meropenem and ertapenem using X-ray crystallography and molecular dynamics (MD) simulations. X-ray crystal structures identify acyl-enzymes in both the {Delta}1-imine and {Delta}2-enamine pyrroline tautomeric forms. MD simulations show the epimeric {Delta}2 tautomers of meropenem and ertapenem to more frequently adopt binding poses competent for hydrolysis, i.e. with an appropriate orientation of the carbapenem 6-hydroxyethyl group and positioning of the water molecule required for deacylation; the results indicate that the {Delta}2 tautomers are preferred for deacylation over the {Delta}1-tautomer. MD simulations based on the crystal structures show that, compared to OXA-48, acyl-enzyme complexes of OXA-519 (a natural OXA-48 variant with a single Val120Leu substitution adjacent to the catalytic general base) more frequently sampled conformations favouring hydrolysis, or formation of the alternative {beta}-lactone deacylation product. MD simulations of complexes derived from quantum mechanics/molecular mechanics (QM/MM) simulations show the meropenem-derived {beta}-lactone product is better retained in the OXA-48 active site than hydrolysed meropenem, consistent with reversible {beta}-lactone formation. Overall, our results demonstrate how acyl-enzyme tautomerisation, dynamics and hydration collectively modulate degradation of 1{beta}-methyl carbapenems by class D {beta}-lactamases of the OXA-48 group, and how subtle changes in active site structure potentiate such effects in the OXA-519 variant.

biochemistry↗

bioRxiv2025

Time-resolved ligand dynamics revealed in a β-lactamase using room-temperature serial crystallography

{beta}-Lactamases catalyze {beta}-lactam antibiotic hydrolysis and are important contributors to bacterial antimicrobial resistance; {beta}-lactamase inhibitors are widely used to overcome {beta}-lactamase-mediated antibiotic resistance. Nucleophilic serine {beta}-lactamases (SBLs) react with their substrates and clinically available inhibitors via a covalent reaction to give complexes which can undergo further reaction. Using room temperature drop on fixed target serial crystallography, where ligands are rapidly mixed with microcrystals, and classical single-crystal crystallography at cryogenic temperatures, we investigate the reversible covalent reaction of the SBL CTX-M-15 with the diazobicyclooctane inhibitor avibactam. We observe avibactam covalently reacted (ring-opened) with the nucleophilic Ser70, at timepoints from 80 ms to minutes (room temperature) and hours (100 K). These crystallographic data reveal time-dependent movement of the avibactam carbamoyl complex, from 1.3 s onwards, that has implications for the 5-exo-trig recyclization mechanism that determines inhibitor reformation. Combined with molecular dynamics simulations and quantum mechanics calculations at the density functional theory level, the results show that in the first seconds of the reaction the avibactam N-sulfate nitrogen is poorly positioned for recyclization. This subsequently equilibrates after 10 s to a stable endpoint that is in a conformation potentially primed to initiate recyclization through attack of the N-sulfate nitrogen on the carbamoyl carbon. These results further demonstrate the capacity of room-temperature serial crystallography to capture time-resolved changes in ligand conformation at an enzyme active site, complementing discrete classical cryo-crystallography. These data inform on ligand dynamics and the stereoelectronics of diazobicyclooctane inhibition, aiding drug discovery efforts to develop inhibitors of nucleophilic enzymes.

biochemistry↗

bioRxiv2025

Drop on fixed target reaction initiation approach for serial and time resolved crystallography

We describe the design and implementation of a drop on fixed target method for time-resolved serial crystallography at both synchrotron and XFEL facilities. A piezoelectric droplet dispensing pipette is employed for addition of picolitre volume (40 - 90 pL) aqueous droplets, containing (co-)substrate(s) or ligand(s), onto enzyme microcrystals immobilised on a solid support. The system was tested with various enzyme systems, including lysozyme and two -lactamases, CTX-M-15 and AmpCEC. Mitigation strategies for cross-well contamination, including the implementation of interleaved controls, are described; the overall performance of the system at synchrotron and X-ray free electron laser facilities was evaluated. This drop on fixed target method is a reliable framework for time-resolved crystallography and will improve the consistency of measurements across facilities.

biophysics↗

bioRxiv2025

Hundred-Picolitre Droplet on Demand Tape Drive and XES Prototypes for Time-Resolved Serial Crystallography at VMXi, Diamond Light Source

Time resolved X-ray crystallography is experiencing a resurgence, in part, because of serial methods that readily allow scientists to create stop-motion movies of macromolecular function of photoactivation, enzyme catalysed reactions, and ligand-induced conformational changes triggering further downstream signalling events. While some reactions can be initiated with light, either naturally or using photocaged compounds, a more generally applicable approach is to mix microcrystals with reagents at varying time points prior to exposure to the X-ray beam. A powerful approach has been to combine droplet on demand tape drive sample delivery with X-ray emission spectroscopy (XES) that correlates atomic structure with electronic states of metal ions within the sample. To our knowledge, such a combined methodology has not been deployed previously at a synchrotron beamline and has been restricted to XFELs. Here we describe prototype experiments along the development pathway to a combined droplet on demand diffraction and XES system at the microfocus beamline VMXi at Diamond Light Source. We demonstrate the collection of a high-quality serial diffraction data set from microcrystals within hundreds of picolitre-volume droplets deposited on a moving tape. In separate experiments at VMXi, we collected XES data from microcrystals of a copper enzyme delivered using a high viscosity extruder. Together, these results demonstrate the feasibility of combined droplet on demand serial crystallography and XES experiments using a third-generation synchrotron beamline; project work currently underway at Diamond Light Source. SynopsisWe describe proof of concept experiments towards correlated serial crystallography (SSX) and X-ray emission spectroscopy (XES) from microcrystals at a microfocus synchrotron beamline. A droplet on demand tape drive system delivers microcrystals to the beam within well-separated, hundreds of picolitre-volume droplets while XES allows validation of redox states of metals within protein crystals.

biophysics↗

bioRxiv2025

Mechanistic Basis for Inhibition of the Extended Spectrum Class A b-Lactamase GES-1 by Tazobactam and Enmetazobactam

Expression of {beta}-lactamases is the primary form of {beta}-lactam antibiotic resistance in Gram-negative bacteria. Enmetazobactam is a penicillanic acid sulfone (PAS) that inhibits extended spectrum {beta}-lactamases (ESBLs) by forming an acyl-enzyme complex that eventually breaks down to an irreversible lysinoalanine crosslink. In contrast, enmetazobactam inhibits the class A carbapenemase KPC-2 via an acyl-enzyme that does not lead to lysinoalanine crosslink formation. This difference correlates with greater inhibitory potency of enmetazobactam against class A ESBLs, compared to carbapenemases. The GES enzymes, unlike other class A {beta}-lactamase families, show progression from carbapenem-inhibited to carbapenem-hydrolysing phenotypes through single point mutations. We present crystal structures of GES-1, a globally disseminated ESBL, as the enmetazobactam- and tazobactam-derived acyl-enzymes. The complexes differ in the identities of their respective covalent adducts, with the catalytic Ser70 acylated by a 214 Da enmetazobactam-derived fragment, whereas tazobactam has fragmented to a 70 Da aldehyde. The tautomeric form of the enmetazobactam-derived ligand is verified by high-level QM/MM calculations, revealing the trans-enamine as the most thermodynamically stable tautomer, that adopts an optimised conformation that best matches the experimentally observed electron density appended to the side chain oxygen of Ser70. In contrast to previous findings for the ESBL CTX-M-15, mass spectrometry provides no evidence for lysinoalanine crosslink formation on reaction of GES-1 with (enme)tazobactam, providing further evidence that PAS inhibitors inhibit different class A {beta}-lactamases by different mechanisms. This work reveals new details of the basis for PAS inhibition of diverse {beta}-lactamases, and will guide development of future {beta}-lactamase inhibitors.

biochemistry↗

bioRxiv2024

Electrostatic interactions define nacubactam potency against OXA-48-like β-lactamases

Carbapenemases, {beta}-lactamases hydrolysing carbapenem antibiotics, severely challenge treatment of multi-drug resistant bacterial infections. OXA-48 is among the most widely disseminated carbapenemases in Enterobacterales, consequently new treatment options for OXA-48 producers are urgently required. Development of diazabicyclooctane (DBO) inhibitors to overcome {beta}-lactamase-mediated resistance is one attractive option, due to their efficacy against a wide range of {beta}-lactamases. The DBO avibactam is already licensed for clinical use, with the related compound nacubactam currently in phase III trials for carbapenem-resistant infections. Here we investigate the activities of avibactam and nacubactam towards OXA-48 and two variants, OXA-163 and OXA-405, that contain deletions in the {beta}5 - {beta}6 loop adjacent to the active site and show modified activity towards different {beta}-lactam classes. Compared to avibactam, nacubactam is c. 80-fold less potent towards OXA-48, but this difference is reduced in OXA-163 and OXA-405. Crystal structures of the respective avibactam and nacubactam complexes, and molecular dynamics simulations based upon these, reveal residue Arg214 on the OXA-48 {beta}5 - {beta}6 active-site loop to be electrostatically repelled by nacubactam, but not avibactam, binding. This increases flexibility of the OXA-48 {beta}5 - {beta}6 loop, as well as neighbouring active site loops, in simulations of the OXA-48:nacubactam, compared to the avibactam, complex. Such effects are not observed in simulations of the respective complexes of OXA-163 and OXA-405, which lack Arg214. These data indicate that interactions with Arg214 can determine DBO potency towards OXA-48-like enzymes, and suggest that sequence variation in this {beta}-lactamase family affects reactivity towards inhibitors as well as {beta}-lactam substrates.

biochemistry↗

bioRxiv2024

Dynamical Responses Predict a Distal Site that Modulates Activity in an Antibiotic Resistance Enzyme

{beta}-Lactamases, which hydrolyse {beta}-lactam antibiotics, are key determinants of antibiotic resistance. Predicting the sites and effects of distal mutations in enzymes is challenging. For {beta}-lactamases, the ability to make such predictions would contribute to understanding activity against, and development of, antibiotics and inhibitors to combat resistance. Here, using dynamical non-equilibrium molecular dynamics (D-NEMD) simulations combined with experiments, we demonstrate that intramolecular communication networks differ in three class A SulpHydryl Variant (SHV)-type {beta}-lactamases). Differences in network architecture and correlated motions link to catalytic efficiency and {beta}-lactam substrate spectrum. Further, the simulations identify a distal residue 89 in the clinically important Klebsiella pneumoniae carbapenemase 2 (KPC-2), as a participant in similar networks, suggesting that mutation at this position would modulate enzyme activity. Experimental kinetics, biophysical and structural characterisation of the naturally occurring, but previously biochemically uncharacterised, KPC-2G89D mutant with several antibiotics and inhibitors reveals significant changes in hydrolytic spectrum, specifically reducing activity towards carbapenems without effecting major structural or stability changes. These results show that D-NEMD simulations can predict distal sites where mutation affects enzyme activity. This approach could have broad application in understanding enzyme evolution, and in engineering of natural and de novo enzymes.

biochemistry↗

bioRxiv2023

Harvesting and amplifying gene cassettes confers cross-resistance to critically important antibiotics

Amikacin and piperacillin/tazobactam are frequent antibiotic choices to treat bloodstream infection, which is commonly fatal and most often caused by bacteria from the family Enterobacterales. Here we show that two gene cassettes located side-by-side in and ancestral integron similar to In37 have been "harvested" by insertion sequence IS26 as a transposon that is already globally disseminated among the Enterobacterales. This transposon encodes the enzymes AAC(6)-Ib-cr and OXA-1, reported, respectively, as amikacin and piperacillin/tazobactam resistance mechanisms. However, by studying bloodstream infection isolates from 769 patients from, three hospitals serving a population of 1.5 million people in South West England, we show that increased enzyme production due to mutation in an IS26/In37-derived hybrid promoter or, more commonly, transposon copy number amplification is required to simultaneously remove these two key therapeutic options; in many cases leaving only the last-resort antibiotic, meropenem. These findings may help improve the accuracy of predicting piperacillin/tazobactam treatment failure, allowing stratification of patients to receive meropenem or piperacillin/tazobactam, which may improve outcome and slow the emergence of meropenem resistance.

microbiology↗

bioRxiv2022

Tautomer-specific deacylation and {Omega}-loop flexibility explain carbapenem-hydrolyzing, broad-spectrum activity of the KPC-2 β-lactamase

KPC-2 (Klebsiella pneumoniae carbapenemase-2) is a globally disseminated serine-{beta}-lactamase (SBL) responsible for extensive {beta}-lactam antibiotic resistance in Gram-negative pathogens. SBLs inactivate {beta}-lactams via a mechanism involving a hydrolytically labile covalent acyl-enzyme intermediate. Carbapenems, the most potent {beta}-lactams, evade activity of many SBLs by forming long-lived inhibitory acyl-enzymes; however, carbapenemases such as KPC-2 efficiently catalyze deacylation of carbapenem-derived acyl-enzymes. We present high-resolution (1.25-1.4 [A]) crystal structures of KPC-2 acyl-enzymes with representative penicillins (ampicillin), cephalosporins (cefalothin) and carbapenems (imipenem, meropenem and ertapenem), obtained utilizing an isosteric deacylation-deficient mutant (E166Q). Mobility of the {Omega}-loop (residues 165-170) negatively correlates with antibiotic turnover rates (kcat), highlighting the role of this region in positioning catalytic residues for efficient hydrolysis of different {beta}-lactams. Carbapenem-derived acyl-enzyme structures reveal predominance of the {Delta}1-(2R) imine tautomer, except for the imipenem acyl-enzyme, which is present in dual occupancy in both {Delta}1-(2R) and (2S) configurations. Quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations of deacylation of the KPC-2:meropenem acyl-enzyme, using an adaptive string method (ASM), show that the {Delta}1-(2R) isomer has a 7 kcal/mol higher barrier for the (rate-determining) formation of the tetrahedral deacylation intermediate than the {Delta}2 tautomer. The simulations identify tautomer-specific differences in hydrogen bonding networks involving the carbapenem C-3 carboxylate and the deacylating water, that, together with stabilization by protonated N-4 of accumulating negative charge during oxyanion formation, accelerate deacylation of the {Delta}2-enamine acyl-enzyme compared to the {Delta}1-imine. Taken together, our data show how the flexible {Omega}-loop helps confer broad spectrum activity upon KPC-2, while carbapenemase activity stems from efficient deacylation of the {Delta}2-enamine acyl-enzyme tautomer. Differentiation of the barriers associated with deacylation of these subtly different {beta}-lactam isomers further identifies ASM as a sensitive method for calculation of reaction energetics that can accurately model turnover and, potentially, predict the impact of substrate modifications or point mutations upon activity.

biochemistry↗

bioRxiv2021

Allosteric communication in Class A β-lactamases occurs via Cooperative Coupling of Loop Dynamics

Allosteric effects control protein (e.g. enzyme) activity in ways that are not fully understood. Better understanding of allosteric effects, and tools to identify them, would offer promising alternative strategies to inhibitor development. Through a combination of equilibrium and nonequilibrium molecular dynamics simulations, we identify allosteric effects and communication pathways from two distant ligand binding sites to important active site structural elements that control enzymatic activity in two prototypical class A {beta}-lactamases, TEM-1 and KPC-2. Both of these enzymes are important determinants of antibiotic resistance in widespread bacterial pathogens. The simulations show that the allosteric sites are connected to the active site in both enzymes, (e.g. affecting the conformation of the {Omega}-loop) highlighting how allosteric inhibitors may exert their effects. Nonequilibrium simulations reveal pathways of communication operating over distances of 30 [A] or more. In these identified signaling pathways, the propagation of the signal occurs through cooperative coupling of loop dynamics. Notably, 50% or more clinically relevant amino acid substitutions in each enzyme 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.

biophysics↗