scholarly journals Crystal Structures of KPC-2 β-Lactamase in Complex with 3-Nitrophenyl Boronic Acid and the Penam Sulfone PSR-3-226

2012 ◽  
Vol 56 (5) ◽  
pp. 2713-2718 ◽  
Author(s):  
Wei Ke ◽  
Christopher R. Bethel ◽  
Krisztina M. Papp-Wallace ◽  
Sundar Ram Reddy Pagadala ◽  
Micheal Nottingham ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Boronic Acid ◽  
Carbonyl Oxygen ◽  
Oxyanion Hole ◽  
Covalent Interaction ◽  
Class A ◽  
Inactivation Rate Constant ◽  
Reversible Covalent ◽  
Acid Compound

ABSTRACTClass A carbapenemases are a major threat to the potency of carbapenem antibiotics. A widespread carbapenemase, KPC-2, is not easily inhibited by β-lactamase inhibitors (i.e., clavulanic acid, sulbactam, and tazobactam). To explore different mechanisms of inhibition of KPC-2, we determined the crystal structures of KPC-2 with two β-lactamase inhibitors that follow different inactivation pathways and kinetics. The first complex is that of a small boronic acid compound, 3-nitrophenyl boronic acid (3-NPBA), bound to KPC-2 with 1.62-Å resolution. 3-NPBA demonstrated aKmvalue of 1.0 ± 0.1 μM (mean ± standard error) for KPC-2 and blocks the active site by making a reversible covalent interaction with the catalytic S70 residue. The two boron hydroxyl atoms of 3-NPBA are positioned in the oxyanion hole and the deacylation water pocket, respectively. In addition, the aromatic ring of 3-NPBA provides an edge-to-face interaction with W105 in the active site. The structure of KPC-2 with the penam sulfone PSR-3-226 was determined at 1.26-Å resolution. PSR-3-226 displayed aKmvalue of 3.8 ± 0.4 μM for KPC-2, and the inactivation rate constant (kinact) was 0.034 ± 0.003 s−1. When covalently bound to S70, PSR-3-226 forms atrans-enamine intermediate in the KPC-2 active site. The predominant active site interactions are generated via the carbonyl oxygen, which resides in the oxyanion hole, and the carboxyl moiety of PSR-3-226, which interacts with N132, N170, and E166. 3-NPBA and PSR-3-226 are the first β-lactamase inhibitors to be trapped as an acyl-enzyme complex with KPC-2. The structural and inhibitory insights gained here could aid in the design of potent KPC-2 inhibitors.

scholarly journals Novel Insights into the Mode of Inhibition of Class A SHV-1 β-Lactamases Revealed by Boronic Acid Transition State Inhibitors

2010 ◽  
Vol 55 (1) ◽  
pp. 174-183 ◽  
Author(s):  
Wei Ke ◽  
Jared M. Sampson ◽  
Claudia Ori ◽  
Fabio Prati ◽  
Sarah M. Drawz ◽  
...  
Keyword(s):  
Transition State ◽  
Active Site ◽  
Boronic Acid ◽  
Kinetic Data ◽  
Binding Pocket ◽  
Stacking Interactions ◽  
Relative Resistance ◽  
Class A ◽  
Important Enzyme ◽  
Phenol Moiety

ABSTRACTBoronic acid transition state inhibitors (BATSIs) are potent class A and C β-lactamase inactivators and are of particular interest due to their reversible nature mimicking the transition state. Here, we present structural and kinetic data describing the inhibition of the SHV-1 β-lactamase, a clinically important enzyme found inKlebsiella pneumoniae, by BATSI compounds possessing the R1 side chains of ceftazidime and cefoperazone and designed variants of the latter, compounds 1 and 2. The ceftazidime and cefoperazone BATSI compounds inhibit the SHV-1 β-lactamase with micromolar affinity that is considerably weaker than their inhibition of other β-lactamases. The solved crystal structures of these two BATSIs in complex with SHV-1 reveal a possible reason for SHV-1's relative resistance to inhibition, as the BATSIs adopt a deacylation transition state conformation compared to the usual acylation transition state conformation when complexed to other β-lactamases. Active-site comparison suggests that these conformational differences might be attributed to a subtle shift of residue A237 in SHV-1. The ceftazidime BATSI structure revealed that the carboxyl-dimethyl moiety is positioned in SHV-1's carboxyl binding pocket. In contrast, the cefoperazone BATSI has its R1 group pointing away from the active site such that its phenol moiety moves residue Y105 from the active site via end-on stacking interactions. To work toward improving the affinity of the cefoperazone BATSI, we synthesized two variants in which either one or two extra carbons were added to the phenol linker. Both variants yielded improved affinity against SHV-1, possibly as a consequence of releasing the strain of its interaction with the unusual Y105 conformation.

scholarly journals Insights Into the Inhibition of MOX-1 β-Lactamase by S02030, a Boronic Acid Transition State Inhibitor

2021 ◽  
Vol 12 ◽  
Author(s):  
Tatsuya Ishikawa ◽  
Nayuta Furukawa ◽  
Emilia Caselli ◽  
Fabio Prati ◽  
Magdalena A. Taracila ◽  
...  
Keyword(s):  
Transition State ◽  
Active Site ◽  
Boronic Acid ◽  
Laboratory Strain ◽  
Multidrug Resistant ◽  
Mechanisms Of Action ◽  
Gram Negative Bacteria ◽  
Class A ◽  
Fixed Concentration ◽  
Active Site Cavity

The rise of multidrug resistant (MDR) Gram-negative bacteria has accelerated the development of novel inhibitors of class A and C β-lactamases. Presently, the search for novel compounds with new mechanisms of action is a clinical and scientific priority. To this end, we determined the 2.13-Å resolution crystal structure of S02030, a boronic acid transition state inhibitor (BATSI), bound to MOX-1 β-lactamase, a plasmid-borne, expanded-spectrum AmpC β-lactamase (ESAC) and compared this to the previously reported aztreonam (ATM)-bound MOX-1 structure. Superposition of these two complexes shows that S02030 binds in the active-site cavity more deeply than ATM. In contrast, the SO3 interactions and the positional change of the β-strand amino acids from Lys315 to Asn320 were more prominent in the ATM-bound structure. MICs were performed using a fixed concentration of S02030 (4 μg/ml) as a proof of principle. Microbiological evaluation against a laboratory strain of Escherichia coli expressing MOX-1 revealed that MICs against ceftazidime are reduced from 2.0 to 0.12 μg/ml when S02030 is added at a concentration of 4 μg/ml. The IC50 and Ki of S02030 vs. MOX-1 were 1.25 ± 0.34 and 0.56 ± 0.03 μM, respectively. Monobactams such as ATM can serve as informative templates for design of mechanism-based inhibitors such as S02030 against ESAC β-lactamases.

scholarly journals Ligand-Dependent Disorder of the Ω Loop Observed in Extended-Spectrum SHV-Type β-Lactamase

2011 ◽  
Vol 55 (5) ◽  
pp. 2303-2309 ◽  
Author(s):  
Jared M. Sampson ◽  
Wei Ke ◽  
Christopher R. Bethel ◽  
S. R. R. Pagadala ◽  
Michael D. Nottingham ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Gram Negative Bacteria ◽  
Structural Alterations ◽  
Critical Amino Acid ◽  
Class A ◽  
Extended Spectrum ◽  
Loop Flexibility ◽  
Bacteria Resistance ◽  
Lactamase Inhibitor

ABSTRACTAmong Gram-negative bacteria, resistance to β-lactams is mediated primarily by β-lactamases (EC 3.2.6.5), periplasmic enzymes that inactivate β-lactam antibiotics. Substitutions at critical amino acid positions in the class A β-lactamase families result in enzymes that can hydrolyze extended-spectrum cephalosporins, thus demonstrating an “extended-spectrum” β-lactamase (ESBL) phenotype. Using SHV ESBLs with substitutions in the Ω loop (R164H and R164S) as target enzymes to understand this enhanced biochemical capability and to serve as a basis for novel β-lactamase inhibitor development, we determined the spectra of activity and crystal structures of these variants. We also studied the inactivation of the R164H and R164S mutants with tazobactam and SA2-13, a unique β-lactamase inhibitor that undergoes a distinctive reaction chemistry in the active site. We noted that the reducedKivalues for the R164H and R164S mutants with SA2-13 are comparable to those with tazobactam (submicromolar). The apo enzyme crystal structures of the R164H and R164S SHV variants revealed an ordered Ω loop architecture that became disordered when SA2-13 was bound. Important structural alterations that result from the binding of SA2-13 explain the enhanced susceptibility of these ESBL enzymes to this inhibitor and highlight ligand-dependent Ω loop flexibility as a mechanism for accommodating and hydrolyzing β-lactam substrates.

Theoretical Studies of the Acid-Base Equilibria in a Model Active Site of the Human 20S Proteasome

2020 ◽  
Author(s):  
Jon Uranga ◽  
Lukas Hasecke ◽  
Jonny Proppe ◽  
Jan Fingerhut ◽  
Ricardo A. Mata
Keyword(s):  
Active Site ◽  
Boronic Acid ◽  
Computational Study ◽  
Ubiquitin Proteasome System ◽  
Bayesian Optimization ◽  
Inhibition Mechanism ◽  
20S Proteasome ◽  
Acid Base ◽  
Ubiquitin Proteasome ◽  
Infection Cycles

The 20S Proteasome is a macromolecule responsible for the chemical step in the ubiquitin-proteasome system of degrading unnecessary and unused proteins of the cell. It plays a central role both in the rapid growth of cancer cells as well as in viral infection cycles. Herein, we present a computational study of the acid-base equilibria in an active site of the human proteasome, an aspect which is often neglected despite the crucial role protons play in the catalysis. As example substrates, we take the inhibition by epoxy and boronic acid containing warheads. We have combined cluster quantum mechanical calculations, replica exchange molecular dynamics and Bayesian optimization of non-bonded potential terms in the inhibitors. In relation to the latter, we propose an easily scalable approach to the reevaluation of non-bonded potentials making use of QM/MM dynamics information. Our results show that coupled acid-base equilibria need to be considered when modeling the inhibition mechanism. The coupling between a neighboring lysine and the reacting threonine is not affected by the presence of the inhibitor.

scholarly journals Crystal structures of non-oxidative decarboxylases reveal a new mechanism of action with a catalytic dyad and structural twists

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Matthias Zeug ◽  
Nebojsa Markovic ◽  
Cristina V. Iancu ◽  
Joanna Tripp ◽  
Mislav Oreb ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Enzyme Engineering ◽  
Base Catalysis ◽  
Oxidative Decarboxylation ◽  
Transition State Stabilization ◽  
Major Bottleneck ◽  
Hydroxybenzoic Acids ◽  
Potassium Binding ◽  
Β Sheet

AbstractHydroxybenzoic acids, like gallic acid and protocatechuic acid, are highly abundant natural compounds. In biotechnology, they serve as critical precursors for various molecules in heterologous production pathways, but a major bottleneck is these acids’ non-oxidative decarboxylation to hydroxybenzenes. Optimizing this step by pathway and enzyme engineering is tedious, partly because of the complicating cofactor dependencies of the commonly used prFMN-dependent decarboxylases. Here, we report the crystal structures (1.5–1.9 Å) of two homologous fungal decarboxylases, AGDC1 from Arxula adenivorans, and PPP2 from Madurella mycetomatis. Remarkably, both decarboxylases are cofactor independent and are superior to prFMN-dependent decarboxylases when heterologously expressed in Saccharomyces cerevisiae. The organization of their active site, together with mutational studies, suggests a novel decarboxylation mechanism that combines acid–base catalysis and transition state stabilization. Both enzymes are trimers, with a central potassium binding site. In each monomer, potassium introduces a local twist in a β-sheet close to the active site, which primes the critical H86-D40 dyad for catalysis. A conserved pair of tryptophans, W35 and W61, acts like a clamp that destabilizes the substrate by twisting its carboxyl group relative to the phenol moiety. These findings reveal AGDC1 and PPP2 as founding members of a so far overlooked group of cofactor independent decarboxylases and suggest strategies to engineer their unique chemistry for a wide variety of biotechnological applications.

scholarly journals Structure and Molecular Recognition Mechanism of IMP-13 Metallo-β-Lactamase

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Charlotte A. Softley ◽  
Krzysztof M. Zak ◽  
Mark J. Bostock ◽  
Roberto Fino ◽  
Richard Xu Zhou ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Rational Drug Design ◽  
Tryptophan Residue ◽  
Resonance Spectroscopy ◽  
Global Public Health ◽  
Recognition Mechanism ◽  
Gram Negative ◽  
Content Type ◽  
Locking Mechanism

ABSTRACT Multidrug resistance among Gram-negative bacteria is a major global public health threat. Metallo-β-lactamases (MBLs) target the most widely used antibiotic class, the β-lactams, including the most recent generation of carbapenems. Interspecies spread renders these enzymes a serious clinical threat, and there are no clinically available inhibitors. We present the crystal structures of IMP-13, a structurally uncharacterized MBL from the Gram-negative bacterium Pseudomonas aeruginosa found in clinical outbreaks globally, and characterize the binding using solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations. The crystal structures of apo IMP-13 and IMP-13 bound to four clinically relevant carbapenem antibiotics (doripenem, ertapenem, imipenem, and meropenem) are presented. Active-site plasticity and the active-site loop, where a tryptophan residue stabilizes the antibiotic core scaffold, are essential to the substrate-binding mechanism. The conserved carbapenem scaffold plays the most significant role in IMP-13 binding, explaining the broad substrate specificity. The observed plasticity and substrate-locking mechanism provide opportunities for rational drug design of novel metallo-β-lactamase inhibitors, essential in the fight against antibiotic resistance.

Sign in / Sign up

Export Citation Format

Share Document