Structures of FOX-4 Cephamycinase in Complex with Transition-State Analog Inhibitors (original) (raw)
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FOX-4 cephamycinase: an analysis of structure and function
Antimicrobial Agents and Chemotherapy, 2015
Class C β-lactamases poorly hydrolyze cephamycins (e.g., cefoxitin, cefotetan, and moxalactam). In the past 2 decades, a new family of plasmid-based AmpC β-lactamases conferring resistance to cefoxitin, the FOX family, has grown to include nine unique members descended from theAeromonas caviaechromosomal AmpC. To understand the basis for the unique cephamycinase activity in the FOX family, we determined the first X-ray crystal structures of FOX-4, apo enzyme and the acyl-enzyme with its namesake compound, cefoxitin, using the Y150F deacylation-deficient variant. Notably, recombinant expression of N-terminally tagged FOX-4 also yielded an inactive adenylylated enzyme form not previously observed in β-lactamases. The posttranslational modification (PTM), which occurs on the active site Ser64, would not seem to provide a selective advantage, yet might present an opportunity for the design of novel antibacterial drugs. Substantial ligand-induced changes in the enzyme are seen in the acy...
Biochemistry, 2014
β-Lactam resistance in Acinetobacter baumannii presents one of the greatest challenges to contemporary antimicrobial chemotherapy. Much of this resistance to cephalosporins derives from the expression of the class C β-lactamase enzymes, known as Acinetobacter-derived cephalosporinases (ADCs). Currently, β-lactamase inhibitors are structurally similar to β-lactam substrates and are not effective inactivators of this class C cephalosporinase. Herein, two boronic acid transition state inhibitors (BATSIs S02030 and SM23) that are chemically distinct from β-lactams were designed and tested for inhibition of ADC enzymes. BATSIs SM23 and S02030 bind with high affinity to ADC-7, a chromosomal cephalosporinase from Acinetobacter baumannii (K i = 21.1 ± 1.9 nM and 44.5 ± 2.2 nM, respectively). The X-ray crystal structures of ADC-7 were determined in both the apo form (1.73 Å resolution) and in complex with S02030 (2.0 Å resolution). In the complex, S02030 makes several canonical interactions: the O1 oxygen of S02030 is bound in the oxyanion hole, and the R1 amide group makes key interactions with conserved residues Asn152 and Gln120. In addition, the carboxylate group of the inhibitor is meant to mimic the C 3 /C 4 carboxylate found in β-lactams. The C 3 /C 4 carboxylate recognition site in class C enzymes is comprised of Asn346 and Arg349 (AmpC numbering), and these residues are conserved in ADC-7. Interestingly, in the ADC-7/S02030 complex, the inhibitor carboxylate group is observed to interact with Arg340, a residue that distinguishes ADC-7 from the related class C enzyme AmpC. A thermodynamic analysis suggests that ΔH driven compounds may be optimized to generate new lead agents. The ADC-7/BATSI complex provides insight into recognition of non-β-lactam inhibitors by ADC enzymes and offers a starting point for the structure-based optimization of this class of novel β-lactamase inhibitors against a key resistance target.
Journal of molecular biology, 2008
Oxyimino-cephalosporin antibiotics, such as ceftazidime, escape the hydrolytic activity of most bacterial beta-lactamases. Their widespread use prompted the emergence of the extended-spectrum beta-lactamases CTX-Ms, which have become highly prevalent. The C7 beta-amino thiazol-oxyimino-amide side chain of ceftazidime has a protective effect against most CTX-M beta-lactamases. However, Asp240Gly CTX-M derivatives demonstrate enhanced hydrolytic activity against this compound. In this work, we present the crystallographic structures of Asp240Gly-harboring enzyme CTX-M-16 in complex with ceftazidime-like glycylboronic acid (resolution 1.80 A) and molecular dynamics simulations of the corresponding acyl-enzyme complex. These experiments revealed breathing motions of CTX-M enzymes and the role of the substitution Asp240Gly in the accommodation of ceftazidime. The substitution Asp240Gly resulted in insertion of the C7 beta side chain of ceftazidime deep in the catalytic pocket and orchest...
Journal of Molecular Biology, 2008
Oxyimino-cephalosporin antibiotics, such as ceftazidime, escape the hydrolytic activity of most bacterial β-lactamases. Their widespread use prompted the emergence of the extended-spectrum β-lactamases CTX-Ms, which have become highly prevalent. The C7 β-amino thiazol-oxyiminoamide side chain of ceftazidime has a protective effect against most CTX-M β-lactamases. However, Asp240Gly CTX-M derivatives demonstrate enhanced hydrolytic activity against this compound. In this work, we present the crystallographic structures of Asp240Gly-harboring enzyme CTX-M-16 in complex with ceftazidime-like glycylboronic acid (resolution 1.80 Å) and molecular dynamics simulations of the corresponding acyl-enzyme complex. These experiments revealed breathing motions of CTX-M enzymes and the role of the substitution Asp240Gly in the accommodation of ceftazidime. The substitution Asp240Gly resulted in insertion of the C7β side chain of ceftazidime deep in the catalytic pocket and orchestrated motions of the active serine Ser70, the β3 strand and the omega loop, which favored the key interactions of the residues 237 and 235 with ceftazidime.
Protein Science, 2011
In Pseudomonas aeruginosa, the chromosomally encoded class C cephalosporinase (AmpC b-lactamase) is often responsible for high-level resistance to b-lactam antibiotics. Despite years of study of these important b-lactamases, knowledge regarding how amino acid sequence dictates function of the AmpC Pseudomonas-derived cephalosporinase (PDC) remains scarce. Insights into structure-function relationships are crucial to the design of both b-lactams and highaffinity inhibitors. In order to understand how PDC recognizes the C 3 /C 4 carboxylate of b-lactams, we first examined a molecular model of a P. aeruginosa AmpC b-lactamase, PDC-3, in complex with a boronate inhibitor that possesses a side chain that mimics the thiazolidine/dihydrothiazine ring and the C 3 /C 4 carboxylate characteristic of b-lactam substrates. We next tested the hypothesis generated by our model, i.e. that more than one amino acid residue is involved in recognition of the C 3 /C 4 b-lactam carboxylate, and engineered alanine variants at three putative carboxylate binding amino acids. Antimicrobial susceptibility testing showed that the PDC-3 blactamase maintains a high level of activity despite the substitution of C 3 /C 4 b-lactam carboxylate recognition residues. Enzyme kinetics were determined for a panel of nine penicillin and cephalosporin analog boronates synthesized as active site probes of the PDC-3 enzyme and the Arg349Ala variant. Our examination of the PDC-3 active site revealed that more than one residue could serve to interact with the C 3 /C 4 carboxylate of the b-lactam. This functional versatility has implications for novel drug design, protein evolution, and resistance profile of this enzyme.
Antimicrobial Agents and Chemotherapy, 2013
ABSTRACTClass C cephalosporinases are a growing threat, and clinical inhibitors of these enzymes are currently unavailable. Previous studies have explored the role of Asn152 in theEscherichia coliAmpC and P99 enzymes and have suggested that interactions between C-6′ or C-7′ substituents on penicillins or cephalosporins and Asn152 are important in determining substrate specificity and enzymatic stability. We sought to characterize the role of Asn152 in the clinically important CMY-2 cephalosporinase with substrates and inhibitors. Mutagenesis of CMY-2 at position 152 yields functional mutants (N152G, -S, and -T) that exhibit improved penicillinase activity and retain cephamycinase activity. We also tested whether the position 152 substitutions would affect the inactivation kinetics of tazobactam, a class A β-lactamase inhibitor within vitroactivity against CMY-2. Using standard assays, we showed that the N152G, -S, and -T variants possessed increased catalytic activity against cefoxi...
Computational analysis of the interactions of a novel cephalosporin derivative with β-lactamases
BMC Structural Biology
Background: One of the main concerns of the modern medicine is the frightening spread of antimicrobial resistance caused mainly by the misuse of antibiotics. The researchers worldwide are actively involved in the search for new classes of antibiotics, and for the modification of known molecules in order to face this threatening problem. We have applied a computational approach to predict the interactions between a new cephalosporin derivative containing an additional β-lactam ring with different substituents, and several serine β-lactamases representative of the different classes of this family of enzymes. Results: The results of the simulations, performed by using a covalent docking approach, has shown that this compound, although able to bind the selected β-lactamases, has a different predicted binding score for the two βlactam rings, suggesting that one of them could be more resistant to the attack of these enzymes and stay available to perform its bactericidal activity. Conclusions: The detailed analysis of the complexes obtained by these simulations suggests possible hints to modulate the affinity of this compound towards these enzymes, in order to develop new derivatives with improved features to escape to degradation.
Antimicrobial Agents and Chemotherapy, 2011
Boronic acid transition state inhibitors (BATSIs) are potent class A and C -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 -lactamase, a clinically important enzyme found in Klebsiella 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 -lactamase with micromolar affinity that is considerably weaker than their inhibition of other -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 -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.
Biochemistry, 2002
The Bacillus licheniformis BS3 -lactamase catalyzes the hydrolysis of the -lactam ring of penicillins, cephalosporins, and related compounds. The production of -lactamases is the most common and thoroughly studied cause of antibiotic resistance. Although they escape the hydrolytic activity of the prototypical Staphylococcus aureus -lactamase, many cephems are good substrates for a large number of -lactamases. However, the introduction of a 7R-methoxy substituent, as in cefoxitin, extends their antibacterial spectrum to many cephalosporin-resistant Gram-negative bacteria. The 7R-methoxy group selectively reduces the hydrolytic action of many -lactamases without having a significant effect on the affinity for the target enzymes, the membrane penicillin-binding proteins. We report here the crystallographic structures of the BS3 enzyme and its acyl-enzyme adduct with cefoxitin at 1.7 Å resolution. The comparison of the two structures reveals a covalent acyl-enzyme adduct with perturbed active site geometry, involving a different conformation of the Ω-loop that bears the essential catalytic Glu166 residue. This deformation is induced by the cefoxitin side chain whose position is constrained by the presence of the R-methoxy group. The hydrolytic water molecule is also removed from the active site by the 7carbonyl of the acyl intermediate. In light of the interactions and steric hindrances in the active site of the structure of the BS3-cefoxitin acyl-enzyme adduct, the crucial role of the conserved Asn132 residue is confirmed and a better understanding of the kinetic results emerges.