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Nature Communications Nov 2022Serial x-ray crystallography can uncover binding events, and subsequent chemical conversions occurring during enzymatic reaction. Here, we reveal the structure, binding...
Serial x-ray crystallography can uncover binding events, and subsequent chemical conversions occurring during enzymatic reaction. Here, we reveal the structure, binding and cleavage of moxalactam antibiotic bound to L1 metallo-β-lactamase (MBL) from Stenotrophomonas maltophilia. Using time-resolved serial synchrotron crystallography, we show the time course of β-lactam hydrolysis and determine ten snapshots (20, 40, 60, 80, 100, 150, 300, 500, 2000 and 4000 ms) at 2.20 Å resolution. The reaction is initiated by laser pulse releasing Zn ions from a UV-labile photocage. Two metal ions bind to the active site, followed by binding of moxalactam and the intact β-lactam ring is observed for 100 ms after photolysis. Cleavage of β-lactam is detected at 150 ms and the ligand is significantly displaced. The reaction product adjusts its conformation reaching steady state at 2000 ms corresponding to the relaxed state of the enzyme. Only small changes are observed in the positions of Zn ions and the active site residues. Mechanistic details captured here can be generalized to other MBLs.
Topics: beta-Lactams; Moxalactam; beta-Lactamases; Crystallography, X-Ray
PubMed: 36450742
DOI: 10.1038/s41467-022-35029-3 -
International Journal of Environmental... Oct 2019Cephalosporins that contain the N-methylthiotetrazole side chain (NMTT-cephalosporin) have been reported to be associated with coagulation-related adverse events;... (Meta-Analysis)
Meta-Analysis
Cephalosporins that contain the N-methylthiotetrazole side chain (NMTT-cephalosporin) have been reported to be associated with coagulation-related adverse events; however, a comprehensive evaluation regarding the association is lacking. A systematic review and meta-analysis were conducted to assess the safety profile of NMTT-cephalosporins with respect to hypoprothrombinemia and bleeding. The MEDLINE, Embase, Cochrane, and RISS databases were systematically searched for clinical studies up to October 2018. The association between NMTT-cephalosporins and hypoprothrombinemia was estimated using an odds ratio (OR) with a 95% confidence interval (CI). A total of 15 studies on cefamandole, cefoperazone, cefotetan, cefmetazole, and moxalactam were identified and included in the meta-analysis. Hypoprothrombinemia (OR 1.676, 95% CI 1.275-2.203) and prothrombin time (PT) prolongation (OR 2.050, 95% CI 1.398-3.005) were significantly associated with NMTT-cephalosporins, whereas bleeding was not (OR 1.359, 95% CI 0.920-2.009). Subgroup analyses revealed that cefoperazone (OR 2.506, 95% CI 1.293-4.860), cefamandole (OR 3.247, 95% CI 1.083-9.733), and moxalactam (OR 3.367, 95% CI 1.725-6.572) were significantly associated with hypoprothrombinemia. An Antimicrobial Stewardship Program led by a multidisciplinary team could play a critical role in monitoring cephalosporin-related hypoprothrombinemia or PT prolongation in patients with underlying clinical conditions at risk for bleeding. The multidisciplinary team could also assist in communicating the potential safety concerns regarding NMTT-cephalosporin use with healthcare professionals to decrease the risk of adverse events.
Topics: Anti-Bacterial Agents; Cephalosporins; Humans; Hypoprothrombinemias; Male
PubMed: 31623191
DOI: 10.3390/ijerph16203937 -
Antimicrobial Agents and Chemotherapy Mar 2023In this study, we aimed to clarify the evolutionary trajectory of a Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) population during...
In this study, we aimed to clarify the evolutionary trajectory of a Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) population during β-lactam antibiotic therapy. Five KPC-Kp isolates were collected from a single patient. Whole-genome sequencing and a comparative genomics analysis were performed on the isolates and all -containing plasmids to predict the population evolution process. Growth competition and experimental evolution assays were conducted to reconstruct the evolutionary trajectory of the KPC-Kp population . Five KPC-Kp isolates (KPJCL-1 to KPJCL-5) were highly homologous, and all harbor an IncFII -containing plasmid (pJCL-1 to pJCL-5). Although the genetic structures of these plasmids were almost identical, distinct copy numbers of the gene were detected. A single copy of was presented in pJCL-1, pJCL-2, and pJCL-5, two copies of ( and ) were presented in pJCL-3, and three copies of were presented in pJCL-4. The -harboring KPJCL-3 isolate presented resistance to ceftazidime-avibactam and cefiderocol. The multicopy strain KPJCL-4 had an elevated ceftazidime-avibactam MIC. The patient had been exposed to ceftazidime, meropenem, and moxalactam, after which KPJCL-3 and KPJCL-4 were isolated, which both showed a significant competitive advantage under antimicrobial pressure . Experimental evolution assays revealed that multicopy-containing cells were increased in the original single-copy -harboring KPJCL-2 population under selection with ceftazidime, meropenem, or moxalactam, generating a low-level ceftazidime-avibactam resistance phenotype. Moreover, mutants with a G532T substitution, G820 to C825 duplication, G532A substitution, G721 to G726 deletion, and A802 to C816 duplication increased in the multicopy-containing KPJCL-4 population, generating high-level ceftazidime-avibactam resistance and reduced cefiderocol susceptibility. Ceftazidime-avibactam and cefiderocol resistance can be selected by β-lactam antibiotics other than ceftazidime-avibactam. Notably, gene amplification and mutation are important in KPC-Kp evolution under antibiotic selection.
Topics: Humans; Ceftazidime; Klebsiella pneumoniae; Meropenem; Klebsiella; Moxalactam; Klebsiella Infections; Anti-Bacterial Agents; Azabicyclo Compounds; beta-Lactamases; Bacterial Proteins; Drug Combinations; Microbial Sensitivity Tests; Cefiderocol
PubMed: 36794957
DOI: 10.1128/aac.01279-22 -
Protein Science : a Publication of the... Mar 2020Emergence of Enterobacteriaceae harboring metallo-β-lactamases (MBL) has raised global threats due to their broad antibiotic resistance profiles and the lack of...
Structural and biochemical analysis of the metallo-β-lactamase L1 from emerging pathogen Stenotrophomonas maltophilia revealed the subtle but distinct di-metal scaffold for catalytic activity.
Emergence of Enterobacteriaceae harboring metallo-β-lactamases (MBL) has raised global threats due to their broad antibiotic resistance profiles and the lack of effective inhibitors against them. We have been studied one of the emerging environmental MBL, the L1 from Stenotrophomonas maltophilia K279a. We determined several crystal structures of L1 complexes with three different classes of β-lactam antibiotics (penicillin G, moxalactam, meropenem, and imipenem), with the inhibitor captopril and different metal ions (Zn , Cd , and Cu ). All hydrolyzed antibiotics and the inhibitor were found binding to two Zn ions mainly through the opened lactam ring and some hydrophobic interactions with the binding pocket atoms. Without a metal ion, the active site is very similarly maintained as that of the native form with two Zn ions, however, the protein does not bind the substrate moxalactam. When two Zn ions were replaced with other metal ions, the same di-metal scaffold was maintained and the added moxalactam was found hydrolyzed in the active site. Differential scanning fluorimetry and isothermal titration calorimetry were used to study thermodynamic properties of L1 MBL compared with New Deli Metallo-β-lactamase-1 (NDM-1). Both enzymes are significantly stabilized by Zn and other divalent metals but showed different dependency. These studies also suggest that moxalactam and its hydrolyzed form may bind and dissociate with different kinetic modes with or without Zn for each of L1 and NDM-1. Our analysis implicates metal ions, in forming a distinct di-metal scaffold, which is central to the enzyme stability, promiscuous substrate binding and versatile catalytic activity. STATEMENT: The L1 metallo-β-lactamase from an environmental multidrug-resistant opportunistic pathogen Stenotrophomonas maltophilia K279a has been studied by determining 3D structures of L1 enzyme in the complexes with several β-lactam antibiotics and different divalent metals and characterizing its biochemical and ligand binding properties. We found that the two-metal center in the active site is critical in the enzymatic process including antibiotics recognition and binding, which explains the enzyme's activity toward diverse antibiotic substrates. This study provides the critical information for understanding the ligand recognition and for advanced drug development.
Topics: Anti-Bacterial Agents; Binding Sites; Biocatalysis; Lactams; Metals, Heavy; Microbial Sensitivity Tests; Models, Molecular; Stenotrophomonas maltophilia; beta-Lactamase Inhibitors; beta-Lactamases
PubMed: 31846104
DOI: 10.1002/pro.3804 -
PloS One 2020β-Lactam antibiotic detection has significant implications in food safety control, environmental monitoring and pharmacokinetics study. Here, we report the development...
β-Lactam antibiotic detection has significant implications in food safety control, environmental monitoring and pharmacokinetics study. Here, we report the development of two BADAN-conjugated β-lactamases, E166Cb and E166Cb/N170Q, as sensitive biosensors for β-lactam antibiotic detection. These biosensors were constructed by coupling an environment-sensitive BADAN probe onto location 166 at the active site of the PenP β-lactamase E166C and E166C/N170Q mutants. They gave fluorescence turn-on signals in response to β-lactam antibiotics. Molecular dynamics simulation of E166Cb suggested that the turn-on signal might be attributed to a polarity change of the microenvironment of BADAN and the removal of the fluorescence quenching effect on BADAN exerted by a nearby Tyr-105 upon the antibiotic binding. In the detection of four β-lactams (penicillin G, penicillin V, cefotaxime and moxalactam), both E166Cb and E166Cb/N170Q delivered signal outputs in an antibiotic-concentration dependent manner with a dynamic range spanning from 10 nM to 1 μM. Compared to E166Cb, E166Cb/N170Q generally exhibited more stable signals owing to its higher deficiency in hydrolyzing the antibiotic analyte. The overall biosensor performance of E166Cb and E166Cb/N170Q was comparable to that of their respective fluorescein-modified counterparts, E166Cf and E166Cf/N170Q. But comparatively, the BADAN-conjugated enzymes showed a higher sensitivity, displayed a faster response in detecting moxalactam and a more stable fluorescence signals towards penicillin G. This study illustrates the potential of BADAN-conjugated β-lactamases as biosensing devices for β-lactam antibiotics.
Topics: 2-Naphthylamine; Anti-Bacterial Agents; Biosensing Techniques; Enzymes, Immobilized; Molecular Dynamics Simulation; beta-Lactamases; beta-Lactams
PubMed: 33125437
DOI: 10.1371/journal.pone.0241594 -
Journal of Medicinal Chemistry Nov 2019Small-molecule aggregates are a leading cause of artifacts in early drug discovery, but little is known about their interactions with proteins, nor why some proteins are...
Small-molecule aggregates are a leading cause of artifacts in early drug discovery, but little is known about their interactions with proteins, nor why some proteins are more susceptible to inhibition than others. A possible reason for this apparent selectivity is that aggregation-based inhibition, as a stoichiometric process, is sensitive to protein concentration, which varies across assays. Alternatively, local protein unfolding by aggregates may lead to selectivity since stability varies among proteins. To deconvolute these effects, we used differentially stable point mutants of a single protein, TEM-1 β-lactamase. Broadly, destabilized mutants had higher affinities for and were more potently inhibited by aggregates versus more stable variants. The addition of the irreversible inhibitor moxalactam destabilized several mutants, and these typically bound tighter to a colloidal particle, while the only mutant it stabilized bound weaker. These results suggest that less-stable enzymes are more easily sequestered and inhibited by colloidal aggregates.
Topics: Enzyme Inhibitors; Mutation; Protein Stability; Thermodynamics; beta-Lactamases
PubMed: 31589047
DOI: 10.1021/acs.jmedchem.9b01019 -
The Journal of Veterinary Medical... Jun 2023The susceptibility of 218 extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae isolates from companion animals to three cephamycins (cefmetazole, flomoxef,...
In vitro efficacy of cephamycins against multiple extended-spectrum β-lactamase-producing Klebsiella pneumoniae, Proteus mirabilis, and Enterobacter cloacae isolates from dogs and cats.
The susceptibility of 218 extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae isolates from companion animals to three cephamycins (cefmetazole, flomoxef, and latamoxef) was investigated. Phenotypic testing found 8 of 120 Klebsiella pneumoniae (KP) and 15 of 69 Enterobacter cloacae (EC) isolates were ESBL and AmpC β-lactamase (ABL) co-producers. Isolates of KP, Proteus mirabilis, and EC that only produced ESBL exhibited susceptibility rates to cefmetazole (95.5%, 82.7%, and 9.3%), flomoxef (99.1%, 96.6%, and 74.0%), and latamoxef (99.1%, 100%, and 100%), respectively. Notably, isolates of KP and EC co-producing ESBL and ABL had significantly lower susceptibility rates to the studied drugs when compared with only ESBL producers. This implies that the in vitro activity of cephamycins against ESBL-producing bacteria can differ depending on ABL production and bacterial species.
Topics: Cats; Dogs; Animals; Klebsiella pneumoniae; Proteus mirabilis; Anti-Bacterial Agents; Enterobacter cloacae; Cefmetazole; Cephamycins; Moxalactam; Cat Diseases; Dog Diseases; Enterobacteriaceae; beta-Lactamases; Microbial Sensitivity Tests
PubMed: 37150609
DOI: 10.1292/jvms.23-0052 -
Emerging Microbes & Infections 2020(), especially methicillin-resistant (MRSA), is a major cause of pneumonia, resulting in severe morbidity and mortality in adults and children. Sortase A (SrtA), which...
(), especially methicillin-resistant (MRSA), is a major cause of pneumonia, resulting in severe morbidity and mortality in adults and children. Sortase A (SrtA), which mediates the anchoring of cell surface proteins in the cell wall, is an important virulence factor of . Here, we found that salvianolic acid A (Sal A), which is a natural product that does not affect the growth of , could inhibit SrtA activity (IC = 5.75 μg/ml) and repress the adhesion of bacteria to fibrinogen, the anchoring of protein A to cell wall, the biofilm formation, and the ability of to invade A549 cells. Furthermore, studies demonstrated that Sal A treatment reduced inflammation and protected mice against lethal pneumonia caused by MRSA. More significantly, full protection (a survival rate of 100%) was achieved when Sal A was administered in combination with latamoxef. Together, these results indicate that Sal A could be developed into a promising therapeutic drug to combat MRSA infections while limiting resistance development.
Topics: Aminoacyltransferases; Animals; Bacterial Proteins; Biofilms; Caffeic Acids; Cysteine Endopeptidases; Drug Synergism; Female; Humans; Lactates; Methicillin-Resistant Staphylococcus aureus; Mice; Mice, Inbred C57BL; Moxalactam; Pneumonia; Staphylococcal Infections
PubMed: 31969071
DOI: 10.1080/22221751.2020.1711817