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International Journal of Molecular... Dec 2022The colibactin island () of formed by 19 genes (55-Kb), encodes non-ribosomal peptide (NRP) and polyketide (PK) synthases, which allow the synthesis of colibactin, a...
The colibactin island () of formed by 19 genes (55-Kb), encodes non-ribosomal peptide (NRP) and polyketide (PK) synthases, which allow the synthesis of colibactin, a suspected hybrid PK-NRP compound that causes damage to DNA in eukaryotic cells. The , an unusual essential gene, is found in the operon structure with the gene in the -encoded machinery. Interestingly, the gene has been annotated as a β-lactamase but no previous study has reported its β-lactamase characteristics. In this study, we (i) investigated the β-lactamase properties of the gene in silico by analysing its phylogenetic relationship with bacterial β-lactamase and peptidase enzymes, (ii) compared its three-dimensional (3D) protein structure with those of bacterial β-lactamase proteins using the Phyr2 database and PyMOL software, and (iii) evaluated in vitro its putative enzymatic activities, including β-lactamase, nuclease, and ribonuclease using protein expression and purification from an BL21 strain. In this study, we reveal a structural configuration of toxin/antitoxin systems in this island. Thus, similar to the toxin/antitoxin systems, the role of the gene within the -island gene group appears as an antitoxin, insofar as it is responsible for the activation of the toxin, which is colibactin. In silico, our analyses revealed that ClbP belonged to the superfamily of β-lactamase, class C. Furthermore, in vitro we were unable to demonstrate its β-lactamase activity, likely due to the fact that the gene requires co-expression with other genes, such as the genes present in the -island (19 genes). More research is needed to better understand its actions, particularly with regards to antibiotics, and to discover whether it has any additional functions due to the importance of this gene and its toxicity.
Topics: Escherichia coli; beta-Lactamases; Genes, vif; Phylogeny; Escherichia coli Proteins; Peptide Hydrolases
PubMed: 36555283
DOI: 10.3390/ijms232415642 -
Current Protein & Peptide Science Oct 2009The discovery that the mechanism of beta-lactam hydrolysis catalyzed by the class A (active site serine-dependent) beta-lactamases proceeds via an acyl-enzyme... (Review)
Review
The discovery that the mechanism of beta-lactam hydrolysis catalyzed by the class A (active site serine-dependent) beta-lactamases proceeds via an acyl-enzyme intermediate was made thirty years ago. Since this discovery, the active site circumstance that enables acylation of the active site serine and further enables hydrolytic deacylation of the acyl-serine intermediate, has received extraordinary scrutiny. The justification for this scrutiny is the direct relevance of the beta-lactamases to the manifestation of bacterial resistance to the beta-lactam antibiotics, and the subsequent (to the discovery of the beta-lactamase acyl-enzyme) recognition of the direct evolutionary relationship between the serine beta-lactamase acyl-enzyme, and the penicillin binding protein acyl-enzyme that is key to beta-lactam antibiotic activity. This short review describes the early events leading to the recognition that serine beta-lactamase catalysis proceeds via an acyl-enzyme intermediate, and summarizes several of the key mechanistic studies--including infrared spectroscopy, cryoenzymology, beta-lactam design, and x-ray crystallography--that have been exploited to understand this pivotal catalytic intermediate.
Topics: Bacterial Infections; Binding Sites; Catalysis; Catalytic Domain; Chemistry, Pharmaceutical; Crystallography, X-Ray; Humans; Hydrolysis; Models, Chemical; Penicillins; Protein Conformation; Serine; Spectrophotometry, Infrared; beta-Lactamases; beta-Lactams
PubMed: 19538154
DOI: 10.2174/138920309789351967 -
FEMS Microbiology Reviews Jul 2000Antibacterial chemotherapy is particularly striking in the family of penicillins and cephalosporins. Over 40 structurally different beta-lactam molecules are available... (Review)
Review
Antibacterial chemotherapy is particularly striking in the family of penicillins and cephalosporins. Over 40 structurally different beta-lactam molecules are available in 73 formulations and the majority of them are currently prescribed for medical use in hospitals. beta-Lactams are well tolerated by humans with few side effects. They interact very specifically with their bacterial target, the D-alanyl-D-alanine carboxypeptidase-transpeptidase usually referred to as DD-peptidase. The outstanding number of beta-lactamases produced by bacteria represent a serious threat to the clinical utility of beta-lactams. The discovery of beta-lactamase inhibitors was thought to solve, in part, the problem of resistance. Unfortunately, bacteria have evolved new mechanisms of resistance to overcome the inhibitory effects of beta-lactamase inactivators. Here, we summarize the diversified mechanistic features of class A beta-lactamases interactions with mechanism-based inhibitors using available microbiological, kinetic and structural data for the prototype TEM beta-lactamases. A brief historical overview of the strategies developed to counteract beta-lactamases will be presented followed by a short description of the chemical events which lead to the inactivation of TEM beta-lactamase by inhibitors from different classes. Finally, an update on the clinical prevalence of natural and inhibitor-resistant enzyme mutants, the total chemical synthesis to design and synthesize a new structure and produced a broad spectrum beta-lactamase inhibitor that mimics the beta-lactam ring, but does not contain it is discussed.
Topics: Anti-Bacterial Agents; Enzyme Inhibitors; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Humans; beta-Lactam Resistance; beta-Lactamase Inhibitors; beta-Lactamases; beta-Lactams
PubMed: 10841972
DOI: 10.1111/j.1574-6976.2000.tb00541.x -
Medecine Et Maladies Infectieuses Feb 2014Bacterial resistance to antibiotics has become a major source of concern for public health. Enterobacteriaceae are among the most common human pathogens, causing... (Review)
Review
Bacterial resistance to antibiotics has become a major source of concern for public health. Enterobacteriaceae are among the most common human pathogens, causing community-acquired as well as hospital-acquired infections. Carbapenem-resistant Enterobacteriaceae have been increasingly reported worldwide since their first identification more than 20 years ago. Three main classes of carbapenemases have been identified: Ambler class A beta-lactamase (KPC), class B (metallo-enzymes), and class D (OXA-48 type). Klebsiella pneumoniae carbapenemases (KPC) was first reported in the United States in the late 1990s and since then worldwide, with a marked endemicity in the United States, Greece, and now Italy. Carbapenemase NDM-1 (New Delhi metallo-beta-lactamase-1) is one of the most recently reported metallo-enzymes. It has spread widely in the Indian sub-continent and now worldwide. Carbapenemases of the oxacillinase-48 type (OXA-48) have been identified mostly in Mediterranean and southern European countries with a rapid spread. An early and quick identification of carbapenemase-producing infected patients, but also of carriers, is mandatory to prevent the spread of these highly resistant pathogens. The early identification of carriers and implementing of cohorting strategies is the only means to prevent nosocomial outbreaks caused by carbapenemase, with very few, if any, therapeutic options.
Topics: Bacterial Proteins; Carbapenems; Drug Resistance, Bacterial; Enterobacteriaceae; Humans; Klebsiella pneumoniae; Public Health; United States; beta-Lactamases
PubMed: 24360201
DOI: 10.1016/j.medmal.2013.11.007 -
Antimicrobial Agents and Chemotherapy Jul 2015β-Lactamase enzymes (EC 3.5.2.6) are a significant threat to the continued use of β-lactam antibiotics to treat infections. A novel non-β-lactam β-lactamase...
β-Lactamase enzymes (EC 3.5.2.6) are a significant threat to the continued use of β-lactam antibiotics to treat infections. A novel non-β-lactam β-lactamase inhibitor with activity against many class A and C and some class D β-lactamase variants, avibactam, is now available in the clinic in partnership with ceftazidime. Here, we explored the activity of avibactam against a variety of characterized isogenic laboratory constructs of β-lactamase inhibitor-resistant variants of the class A enzyme SHV (M69I/L/V, S130G, K234R, R244S, and N276D). We discovered that the S130G variant of SHV-1 shows the most significant resistance to inhibition by avibactam, based on both microbiological and biochemical characterizations. Using a constant concentration of 4 mg/liter of avibactam as a β-lactamase inhibitor in combination with ampicillin, the MIC increased from 1 mg/liter for blaSHV-1 to 256 mg/liter for blaSHV S130G expressed in Escherichia coli DH10B. At steady state, the k2/K value of the S130G variant when inactivated by avibactam was 1.3 M(-1) s(-1), versus 60,300 M(-1) s(-1) for the SHV-1 β-lactamase. Under timed inactivation conditions, we found that an approximately 1,700-fold-higher avibactam concentration was required to inhibit SHV S130G than the concentration that inhibited SHV-1. Molecular modeling suggested that the positioning of amino acids in the active site of SHV may result in an alternative pathway of inactivation when complexed with avibactam, compared to the structure of CTX-M-15-avibactam, and that S130 plays a role in the acylation of avibactam as a general acid/base. In addition, S130 may play a role in recyclization. As a result, we advance that the lack of a hydroxyl group at position 130 in the S130G variant of SHV-1 substantially slows carbamylation of the β-lactamase by avibactam by (i) removing an important proton acceptor and donator in catalysis and (ii) decreasing the number of H bonds. In addition, recyclization is most likely also slow due to the lack of a general base to initiate the process. Considering other inhibitor-resistant mechanisms among class A β-lactamases, S130 may be the most important amino acid for the inhibition of class A β-lactamases, perhaps even for the novel diazabicyclooctane class of β-lactamase inhibitors.
Topics: Acylation; Ampicillin; Anti-Bacterial Agents; Azabicyclo Compounds; Catalytic Domain; Ceftazidime; Drug Combinations; Drug Resistance, Bacterial; Escherichia coli; Microbial Sensitivity Tests; Models, Molecular; beta-Lactamase Inhibitors; beta-Lactamases
PubMed: 25691639
DOI: 10.1128/AAC.04405-14 -
Clinical Microbiology and Infection :... Jun 2002Carbapenemases may be defined as beta-lactamases that significantly hydrolyze at least imipenem or/and meropenem. Carbapenemases involved in acquired resistance are of... (Comparative Study)
Comparative Study Review
Carbapenemases may be defined as beta-lactamases that significantly hydrolyze at least imipenem or/and meropenem. Carbapenemases involved in acquired resistance are of Ambler molecular classes A, B, and D. Class A, clavulanic acid-inhibited carbapenemases are rare. They are either chromosomally encoded (NMC-A, Sme-1 to Sme-3, IMI-1) in Enterobacter cloacae and Serratia marcescens, or plasmid encoded, such as KPC-1 in Klebsiella pneumoniae and GES-2 in Pseudomonas aeruginosa, the latter being a point-mutant of the clavulanic acid-inhibited extended-spectrum beta-lactamase GES-1. The class B enzymes are the most clinically significant carbapenemases. They are metalloenzymes of the IMP or VIM series. They have been reported worldwide but mostly from South East Asia and Europe. Metalloenzymes, whose genes are plasmid and integron located, hydrolyze virtually all beta-lactams except aztreonam. Finally, the class D carbapenemases are increasingly reported in Acinetobacter baumannii but compromise imipenem and meropenem susceptibility only marginally. The sources of the acquired carbapenemase genes remain unknown, as does the relative importance of the spread of epidemic strains as opposed to the spread of plasmid- or integron-borne genes. Because most of these carbapenemases confer only reduced susceptibility to carbapenems in Enterobacteriaceae, they may remain underestimated as a consequence of the lack of their detection.
Topics: Amino Acid Sequence; Bacterial Proteins; Carrier Proteins; Cell Cycle Proteins; Clavulanic Acid; Fungal Proteins; Gram-Negative Aerobic Bacteria; Intracellular Signaling Peptides and Proteins; Molecular Sequence Data; Penicillinase; Protein Kinases; Protein Serine-Threonine Kinases; Saccharomyces cerevisiae Proteins; Sequence Alignment; beta-Lactamases
PubMed: 12084099
DOI: 10.1046/j.1469-0691.2002.00401.x -
ChemistryOpen Oct 2020We present a sensitive and rapid screening method for the determination of β-lactamase activity of antibiotic-resistant bacteria, by designing a pH-sensitive...
We present a sensitive and rapid screening method for the determination of β-lactamase activity of antibiotic-resistant bacteria, by designing a pH-sensitive fluorescent dye-doped mesoporous silica nanoparticle encapsulated with penicillin G as a substrate. When penicillin G was hydrolysed by β-lactamase and converted into penicilloic acid, the acidic environment resulted in fluorescence quenching of the dye. The dye-doped mesoporous nanoparticles not only enhanced the β-lactamase-catalyzed reaction rate but also stablized the substrate, penicillin G, which degrades into penicilloic acid in a water solution without β-lactamase. Twentyfive clinical bacterial samples were tested and the antibiotic resistant and susceptible strains were identified. The proposed method may detect the presence of β -lactamases of clinically relevant samples in less than 1 hour. Moreover, the detection limit of β-lactamase activity was as low as 7.8×10 U/mL, which was determined within two hours.
Topics: Biocatalysis; Enzyme Assays; Fluorescent Dyes; Kinetics; Nanoparticles; Penicillin G; Porosity; Time Factors; Water; beta-Lactamases
PubMed: 33117628
DOI: 10.1002/open.202000221 -
Proceedings of the National Academy of... Nov 2021Understanding the functional role of protein-excited states has important implications in protein design and drug discovery. However, because these states are difficult...
Understanding the functional role of protein-excited states has important implications in protein design and drug discovery. However, because these states are difficult to find and study, it is still unclear if excited states simply result from thermal fluctuations and generally detract from function or if these states can actually enhance protein function. To investigate this question, we consider excited states in β-lactamases and particularly a subset of states containing a cryptic pocket which forms under the Ω-loop. Given the known importance of the Ω-loop and the presence of this pocket in at least two homologs, we hypothesized that these excited states enhance enzyme activity. Using thiol-labeling assays to probe Ω-loop pocket dynamics and kinetic assays to probe activity, we find that while this pocket is not completely conserved across β-lactamase homologs, those with the Ω-loop pocket have a higher activity against the substrate benzylpenicillin. We also find that this is true for TEM β-lactamase variants with greater open Ω-loop pocket populations. We further investigate the open population using a combination of NMR chemical exchange saturation transfer experiments and molecular dynamics simulations. To test our understanding of the Ω-loop pocket's functional role, we designed mutations to enhance/suppress pocket opening and observed that benzylpenicillin activity is proportional to the probability of pocket opening in our designed variants. The work described here suggests that excited states containing cryptic pockets can be advantageous for function and may be favored by natural selection, increasing the potential utility of such cryptic pockets as drug targets.
Topics: Binding Sites; Escherichia coli; Escherichia coli Proteins; Molecular Dynamics Simulation; Mutation; Penicillin G; Penicillinase; Protein Conformation; Proteins; beta-Lactamases
PubMed: 34799442
DOI: 10.1073/pnas.2106473118 -
Chemistry (Weinheim An Der Bergstrasse,... Apr 2018The most important resistance mechanism to β-lactam antibiotics involves hydrolysis by two β-lactamase categories: the nucleophilic serine and the...
The most important resistance mechanism to β-lactam antibiotics involves hydrolysis by two β-lactamase categories: the nucleophilic serine and the metallo-β-lactamases (SBLs and MBLs, respectively). Cyclobutanones are hydrolytically stable β-lactam analogues with potential to inhibit both SBLs and MBLs. We describe solution and crystallographic studies on the interaction of a cyclobutanone penem analogue with the clinically important MBL SPM-1. NMR experiments using F-labeled SPM-1 imply the cyclobutanone binds to SPM-1 with micromolar affinity. A crystal structure of the SPM-1:cyclobutanone complex reveals binding of the hydrated cyclobutanone through interactions with one of the zinc ions, stabilisation of the hydrate by hydrogen bonding to zinc-bound water, and hydrophobic contacts with aromatic residues. NMR analyses using a C-labeled cyclobutanone support assignment of the bound species as the hydrated ketone. The results inform on how MBLs bind substrates and stabilize tetrahedral intermediates. They support further investigations on the use of transition-state and/or intermediate analogues as inhibitors of all β-lactamase classes.
Topics: Catalysis; Cyclobutanes; Meropenem; Molecular Mimicry; Nuclear Magnetic Resonance, Biomolecular; Thienamycins; beta-Lactamase Inhibitors; beta-Lactamases; beta-Lactams
PubMed: 29250863
DOI: 10.1002/chem.201705886 -
Journal of Biochemistry Dec 2022IMP-type metallo-β-lactamases confer resistance to carbapenems and a broad spectrum of β-lactam antibiotics. IMP-6 and IMP-1 differ by only a point mutation: Ser262 in...
IMP-type metallo-β-lactamases confer resistance to carbapenems and a broad spectrum of β-lactam antibiotics. IMP-6 and IMP-1 differ by only a point mutation: Ser262 in IMP-1 and Gly262 in IMP-6. The kcat/Km values of IMP-1 for imipenem and meropenem are nearly identical; however, for IMP-6, the kcat/Km for meropenem is 7-fold that for imipenem. In clinical practice, this may result in an ineffective therapeutic regimen and, consequently, in treatment failure. Here, we report the crystal structures of IMP-6 and IMP-1 with the same space group and similar cell constants at resolutions of 1.70 and 1.94 Å, respectively. The overall structures of IMP-6 and IMP-1 are similar. However, the loop region (residues 60-66), which participates in substrate binding, is more flexible in IMP-6 than in IMP-1. This difference in flexibility determines the substrate specificity of IMP-type metallo-β-lactamases for imipenem and meropenem. The amino acid at position 262 alters the mobility of His263; this affects the flexibility of the loop via a hydrogen bond with Pro68, which plays the role of a hinge in IMP-type metallo-β-lactamases. The substitution of Pro68 with a glycine elicited an increase in the Km of IMP-6 for imipenem, whereas the affinity for meropenem remained unchanged.
Topics: Meropenem; Substrate Specificity; beta-Lactamases; Imipenem; Carbapenems; Anti-Bacterial Agents; Microbial Sensitivity Tests
PubMed: 36174533
DOI: 10.1093/jb/mvac080