Engineered Mononuclear Variants in Bacillus cereus Metallo-β-lactamase BcII Are Inactive (original) (raw)
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Frontiers in Chemistry
β-Lactams are the most widely employed antibiotics in clinical settings due to their broad efficacy and low toxicity. However, since their first use in the 1940s, resistance to β-lactams has proliferated to the point where multi-drug resistant organisms are now one of the greatest threats to global human health. Many bacteria use β-lactamases to inactivate this class of antibiotics via hydrolysis. Although nucleophilic serine-β-lactamases have long been clinically important, most broad-spectrum β-lactamases employ one or two metal ions (likely Zn2+) in catalysis. To date, potent and clinically useful inhibitors of these metallo-β-lactamases (MBLs) have not been available, exacerbating their negative impact on healthcare. MBLs are categorised into three subgroups: B1, B2, and B3 MBLs, depending on their sequence similarities, active site structures, interactions with metal ions, and substrate preferences. The majority of MBLs associated with the spread of antibiotic resistance belong...
Approaches to the simultaneous inactivation of metallo- and serine-β-lactamases
Bioorganic & Medicinal Chemistry Letters, 2009
A series of cephalosporin-derived reverse hydroxamates and oximes were prepared and evaluated as inhibitors of representative metallo-and serine-β-lactamases. The reverse hydroxamates showed submicromolar inhibition of the GIM-1 metallo-β-lactamase. With respect to interactions with the classes A, C, and D serine β-lactamases, as judged by their correspondingly low Km values, the reverse hydroxamates were recognized in a manner similar to the non-hydroxylated N-H amide side chains of the natural substrates of these enzymes. This indicates that, with respect to recognition in the active site of the serine β-lactamases, the O=C-NR-OH functionality can function as a structural isostere of the O=C-NR-H group, with the NO-H group presumably replacing the amide N-H group as a hydrogen bond donor to the appropriate backbone carbonyl oxygen of the protein. The reverse hydroxamates, however, displayed k cat values up to three orders of magnitude lower than the natural substrates, thus indicating substantial slowing of the hydrolytic action of these serine β-lactamases. Although the degree of inactivation is not yet enough to be clinically useful, these initial results are promising. The substitution of the amide N-H bond by N-OH may represent a useful strategy for the inhibition of other serine hydrolases.
Metallo-β-lactamase structure and function
Annals of the New York Academy of Sciences, 2012
β-lactam antibiotics are the most commonly used antibacterial agents and growing resistance to these drugs is a concern. Metallo-β-lactamases are a diverse set of enzymes that catalyze the hydrolysis of a broad range of β-lactam drugs including carbapenems. This diversity is reflected in the observation that the enzyme mechanisms differ based on whether one or two zincs are bound in the active site which, in turn, is dependent on the subclass of β-lactamase. The dissemination of the genes encoding these enzymes among Gram-negative bacteria has made them an important cause of resistance. In addition, there are currently no clinically available inhibitors to block metallo-β-lactamase action. This review summarizes the numerous studies that have yielded insights into the structure, function, and mechanism of action of these enzymes.
Journal of Biological Chemistry, 2002
The L1 metallo--lactamase from Stenotrophomonas maltophilia is unique among this class of enzymes because it is tetrameric. Previous work predicted that the two regions of important intersubunit interaction were the residue Met-140 and the N-terminal extensions of each subunit. The N-terminal extension was also implicated in -lactam binding. Mutation of methionine 140 to aspartic acid results in a monomeric L1 -lactamase with a greatly altered substrate specificity profile. A 20-amino acid N-terminal deletion mutant enzyme (N-Del) could be isolated in a tetrameric form but demonstrated greatly reduced rates of -lactam hydrolysis and different substrate profiles compared with that of the parent enzyme. Specific site-directed mutations of individual N terminus residues were made (Y11S, W17S, and a double mutant L5A/L8A). All N-terminal mutant enzymes were tetramers and all showed higher K m values for ampicillin and nitrocefin, hydrolyzed ceftazidime poorly, and hydrolyzed imipenem more efficiently than ampicillin in contrast to wild-type L1. Nitrocefin turnover was significantly increased, probably because of an increased rate of breakdown of the intermediate species due to a lack of stabilizing forces. K m values for monomeric L1 were greatly increased for all antibiotics tested. A model of a highly mobile N-terminal extension in the monomeric enzyme is proposed to explain these findings. Tetrameric L1 shows negative cooperativity, which is not present in either the monomer or N-terminal deletion enzymes, suggesting that the cooperative effect is mediated via N-terminal intersubunit interactions. These data indicate that while the N terminus of L1 is not essential for -lactam hydrolysis, it is clearly important to its activity and substrate specificity. Stenotrophomonas maltophilia is an opportunistic pathogen that is emerging as a significant cause of nosocomial infections, particularly among immunocompromised patients (1, 2). S. maltophilia is inherently resistant to most antibacterial drugs, including most, if not all, -lactams (3, 4).
Structural Aspects for Evolution of β-Lactamases from Penicillin-Binding Proteins
Journal of the American Chemical Society, 2003
Penicillin-binding proteins (PBPs), biosynthetic enzymes of bacterial cell wall assembly, and -lactamases, resistance enzymes to -lactam antibiotics, are related to each other from an evolutionary point of view. Massova and Mobashery (Antimicrob. Agents Chemother. 1998, 42, 1-17) have proposed that for -lactamases to have become effective at their function as antibiotic resistance enzymes, they would have had to undergo structure alterations such that they would not interact with the peptidoglycan, which is the substrate for PBPs. A cephalosporin analogue, 7 -[N-Acetyl-L-alanyl-γ-D-glutamyl-L-lysine]-3-acetoxymethyl-3-cephem-carboxylic acid (compound 6), was conceived and synthesized to test this notion. The X-ray structure of the complex of this cephalosporin bound to the active site of the deacylation-deficient Q120L/Y150E variant of the class C AmpC -lactamase from Escherichia coli was solved at 1.71 Å resolution. This complex revealed that the surface for interaction with the strand of peptidoglycan that acylates the active site, which is present in PBPs, is absent in the -lactamase active site. Furthermore, insertion of a peptide in the -lactamase active site at a location where the second strand of peptidoglycan in some PBPs binds has effectively abolished the possibility for such interaction with the -lactamase. A 2.6 ns dynamics simulation was carried out for the complex, which revealed that the peptidoglycan surrogate (i.e., the active-site-bound ligand) undergoes substantial motion and is not stabilized for binding within the active site. These factors taken together disclose the set of structure modifications in the antibiotic resistance enzyme that prevent it from interacting with the peptidoglycan, en route to achieving catalytic proficiency for their intended function.
PLoS Pathogens, 2014
Pseudomonas aeruginosa is one of the most virulent and resistant non-fermenting Gram-negative pathogens in the clinic. Unfortunately, P. aeruginosa has acquired genes encoding metallo-b-lactamases (MbLs), enzymes able to hydrolyze most blactam antibiotics. SPM-1 is an MbL produced only by P. aeruginosa, while other MbLs are found in different bacteria. Despite similar active sites, the resistance profile of MbLs towards b-lactams changes from one enzyme to the other. SPM-1 is unique among pathogen-associated MbLs in that it contains ''atypical'' second sphere residues (S84, G121). Codon randomization on these positions and further selection of resistance-conferring mutants was performed. MICs, periplasmic enzymatic activity, Zn(II) requirements, and protein stability was assessed. Our results indicated that identity of second sphere residues modulates the substrate preferences and the resistance profile of SPM-1 expressed in P. aeruginosa. The second sphere residues found in wild type SPM-1 give rise to a substrate selectivity that is observed only in the periplasmic environment. These residues also allow SPM-1 to confer resistance in P. aeruginosa under Zn(II)-limiting conditions, such as those expected under infection. By optimizing the catalytic efficiency towards b-lactam antibiotics, the enzyme stability and the Zn(II) binding features, molecular evolution meets the specific needs of a pathogenic bacterial host by means of substitutions outside the active site.
Drifted catalytic properties of β-lactamases due to unconstrained use of antibiotics
Context: Antibiotic resistance is an old problem with new face as the rate of infections due to multidrug resistant bacteria is increasing everyday and the number of new antibiotics to overwhelm the problem is becoming smaller. Major mechanism beneath this growing resistance is concomitant with the changes in β-lactamases catalytic activity and its functional enhancement. Objectives: In β-lactamases secreting clinical isolates at least 10% are extended-spectrum β-lactamases (ESBL) that are not even treatable with β-lactamases inhibitor like clavulanic acids. This implies that the catalytic domains of β-lactamases have been mutated towards higher pathogenicity. The aim of the present study is to define the changes in β-lactamases catalytic efficiency against β-lactam antibiotics and its inhibitors. Materials and Methods: In this research work we have used multiple drug resistant (MDR) strains from surgical site of infections. A rapid method was used for specific detection of bacterial β-lactamases that uses β-lactam antibiotics as substrates. In this, the end products (open beta-lactam ring forms) generated after separately incubating substrates with β-lactamases producing strains. Those end products of antibiotics were highly fluorescent after specific treatment and could be analyzed visually under long-wave UV lamp for efficiency. Results: β-lactamases secreting strains are variably capable of defending β-lactam antibiotics. Interestingly, one of the E. coli strain secretes ESBL, this means that the strain is resistant against clavulanic acid. However, the most fascinating fact of the finding is that ideally the β-lactamases supposed to hydrolyze Penicillin by default but in our isolates, β-lactamases are not able to hydrolyze penicillin instead they hydrolyze amoxicillin, a derivative which replaced clinical use of penicillin. In addition to that we have identified the presence of New Delhi Metalo-betalactamase in one of the clinical isolates. Conclusion: Rate of evolution in microbes is very high. Thus we presume that some of the amino acids in the functional domain of β-lactamases have been changed respective to extinct use of penicillin whereas it is effective against clinically used other beta lactam antibiotics.
Overcoming differences: The catalytic mechanism of metallo-β-lactamases
FEBS letters, 2015
Metallo-β-lactamases are the latest resistance mechanism of pathogenic and opportunistic bacteria against carbapenems, considered as last resort drugs. The worldwide spread of genes coding for these enzymes, together with the lack of a clinically useful inhibitor, have raised a sign of alarm. Inhibitor design has been mostly impeded by the structural diversity of these enzymes. Here we provide a critical review of mechanistic studies of the three known subclasses of metallo-β-lactamases, analyzed at the light of structural and mutagenesis investigations. We propose that these enzymes present a modular structure in their active sites that can be dissected into two halves: one providing the attacking nucleophile, and the second one stabilizing a negatively charged reaction intermediate. These are common mechanistic elements in all metallo-β-lactamases. Nucleophile activation does not necessarily requires a Zn(II) ion, but a Zn(II) center is essential for stabilization of the anionic i...