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Shahriar Mobashery - One of the best experts on this subject based on the ideXlab platform.

  • conformational dynamics in Penicillin Binding Protein 2a of methicillin resistant staphylococcus aureus allosteric communication network and enablement of catalysis
    Journal of the American Chemical Society, 2017
    Co-Authors: Kiran V Mahasenan, Juan A Hermoso, Mayland Chang, Jed F Fisher, Rafael Molina, Renee Bouley, Maria T Batuecas, Shahriar Mobashery
    Abstract:

    The mechanism of the β-lactam antibacterials is the functionally irreversible acylation of the enzymes that catalyze the cross-linking steps in the biosynthesis of their peptidoglycan cell wall. The Gram-positive pathogen Staphylococcus aureus uses one primary resistance mechanism. An enzyme, called Penicillin-Binding Protein 2a (PBP2a), is brought into this biosynthetic pathway to complete the cross-linking. PBP2a effectively discriminates against the β-lactam antibiotics as potential inhibitors, and in favor of the peptidoglycan substrate. The basis for this discrimination is an allosteric site, distal from the active site, that when properly occupied concomitantly opens the gatekeeper residues within the active site and realigns the conformation of key residues to permit catalysis. We address the molecular basis of this regulation using crystallographic studies augmented by computational analyses. The crystal structures of three β-lactams (oxacillin, cefepime, ceftazidime) complexes with PBP2a—each wit...

  • Penicillin Binding Protein 2a of methicillin resistant staphylococcus aureus
    Iubmb Life, 2014
    Co-Authors: Jennifer Fishovitz, Juan A Hermoso, Mayland Chang, Shahriar Mobashery
    Abstract:

    High-level resistance to β-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) is due to expression of Penicillin-Binding Protein 2a (PBP2a), a transpeptidase that catalyzes cell-wall crosslinking in the face of the challenge by β-lactam antibiotics. The activity of this Protein is regulated by allostery at a site 60 A distant from the active site, where crosslinking of cell wall takes place. This review discusses the state of knowledge on this important enzyme of cell-wall biosynthesis in MRSA.

  • a computational evaluation of the mechanism of Penicillin Binding Protein catalyzed cross linking of the bacterial cell wall
    Journal of the American Chemical Society, 2011
    Co-Authors: Qicun Shi, Samy O. Meroueh, Jed F Fisher, Shahriar Mobashery
    Abstract:

    Penicillin-Binding Protein 1b (PBP 1b) of the Gram-positive bacterium Streptococcus pneumoniae catalyzes the cross-linking of adjacent peptidoglycan strands, as a critical event in the biosynthesis of its cell wall. This enzyme is representative of the biosynthetic PBP structures of the β-lactam-recognizing enzyme superfamily and is the target of the β-lactam antibiotics. In the cross-linking reaction, the amide between the -d-Ala-d-Ala dipeptide at the terminus of a peptide stem acts as an acyl donor toward the e-amino group of a lysine found on an adjacent stem. The mechanism of this transpeptidation was evaluated using explicit-solvent molecular dynamics simulations and ONIOM quantum mechanics/molecular mechanics calculations. Sequential acyl transfer occurs to, and then from, the active site serine. The resulting cross-link is predicted to have a cis-amide configuration. The ensuing and energetically favorable cis- to trans-amide isomerization, within the active site, may represent the key event drivi...

  • crystal structures of Penicillin Binding Protein 6 from escherichia coli
    Journal of the American Chemical Society, 2009
    Co-Authors: Yu Chen, Shahriar Mobashery, Weilie Zhang, Qicun Shi, Dusan Hesek, Mijoon Lee, Brian K Shoichet
    Abstract:

    Penicillin-Binding Protein 6 (PBP6) is one of the two main dd-carboxypeptidases in Escherichia coli, which are implicated in maturation of bacterial cell wall and formation of cell shape. Here, we report the first X-ray crystal structures of PBP6, capturing its apo state (2.1 A), an acyl-enzyme intermediate with the antibiotic ampicillin (1.8 A), and for the first time for a PBP, a preacylation complex (a “Michaelis complex”, determined at 1.8 A) with a peptidoglycan substrate fragment containing the full pentapeptide, NAM-(l-Ala-d-isoGlu-l-Lys-d-Ala-d-Ala). These structures illuminate the molecular interactions essential for ligand recognition and catalysis by dd-carboxypeptidases, and suggest a coupling of conformational flexibility of active site loops to the reaction coordinate. The substrate fragment complex structure, in particular, provides templates for models of cell wall recognition by PBPs, as well as substantiating evidence for the molecular mimicry by β-lactam antibiotics of the peptidoglycan...

  • Catalytic mechanism of Penicillin-Binding Protein 5 of Escherichia coli.
    Biochemistry, 2007
    Co-Authors: Weilie Zhang, Samy O. Meroueh, Sergei B. Vakulenko, Shahriar Mobashery
    Abstract:

    Penicillin-Binding Proteins (PBPs) and β-lactamases are members of large families of bacterial enzymes. These enzymes undergo acylation at a serine residue with their respective substrates as the first step in their catalytic events. Penicillin-Binding Protein 5 (PBP 5) of Escherichia coli is known to perform a dd-carboxypeptidase reaction on the bacterial peptidoglycan, the major constituent of the cell wall. The roles of the active site residues Lys47 and Lys213 in the catalytic machinery of PBP 5 have been explored. By a sequence of site-directed mutagenesis and chemical modification, we individually introduced γ-thialysine at each of these positions. The pH dependence of kcat/Km and of kcat for the wild-type PBP 5 and for the two γ-thialysine mutant variants at positions 47 and 213 were evaluated. The pH optimum for the enzyme was at 9.5−10.5. The ascending limb to the pH optimum is due to Lys47; hence, this residue exists in the free-base form for catalysis. The descending limb from the pH optimum is...

Mariana G Pinho - One of the best experts on this subject based on the ideXlab platform.

  • the staphylococcus aureus chaperone prsa is a new auxiliary factor of oxacillin resistance affecting Penicillin Binding Protein 2a
    Antimicrobial Agents and Chemotherapy, 2016
    Co-Authors: Ambre Jousselin, Mariana G Pinho, Caroline Stephanie Manzano, Alexandra Biette, Patricia Reed, Adriana E Rosato, William Kelley, Adriana Maria Renzoni
    Abstract:

    Expression of the methicillin-resistant S. aureus (MRSA) phenotype results from the expression of the extra Penicillin-Binding Protein 2A (PBP2A), which is encoded by mecA and acquired horizontally on part of the SCCmec cassette. PBP2A can catalyze dd-transpeptidation of peptidoglycan (PG) because of its low affinity for β-lactam antibiotics and can functionally cooperate with the PBP2 transglycosylase in the biosynthesis of PG. Here, we focus upon the role of the membrane-bound PrsA foldase Protein as a regulator of β-lactam resistance expression. Deletion of prsA altered oxacillin resistance in three different SCCmec backgrounds and, more importantly, caused a decrease in PBP2A membrane amounts without affecting mecA mRNA levels. The N- and C-terminal domains of PrsA were found to be critical features for PBP2A Protein membrane levels and oxacillin resistance. We propose that PrsA has a role in posttranscriptional maturation of PBP2A, possibly in the export and/or folding of newly synthesized PBP2A. This additional level of control in the expression of the mecA-dependent MRSA phenotype constitutes an opportunity to expand the strategies to design anti-infective agents.

  • recruitment of Penicillin Binding Protein pbp2 to the division site of staphylococcus aureus is dependent on its transpeptidation substrates
    Molecular Microbiology, 2004
    Co-Authors: Mariana G Pinho, Jeffery Errington
    Abstract:

    Summary Staphylococcus aureus Penicillin-Binding Protein PBP2 is an enzyme involved in the last stages of peptidoglycan assembly and is an important player in the mechanism of methicillin resistance of this pathogen. PBP2 localized to the division site but its recruitment to the forming division septum was prevented after acylation by oxacillin. The presence of the antibiotic did not affect FtsZ ring maintenance nor the localization of externalized peptidoglycan precursors. Delocalization of PBP2 was also observed when its pentapeptide substrate was eliminated by addition of d-cycloserine or blocked by addition of vancomycin. Taken together these observations suggest that PBP2 is recruited to the division site by Binding to its substrate, which is localized at that place. In methicillin-resistant S. aureus, addition of oxacillin does not result in delocalization of PBP2 indicating that acylated PBP2 can be maintained in place by functional PBP2A, the central element of this resistance mechanism.

  • complementation of the essential peptidoglycan transpeptidase function of Penicillin Binding Protein 2 pbp2 by the drug resistance Protein pbp2a in staphylococcus aureus
    Journal of Bacteriology, 2001
    Co-Authors: Mariana G Pinho, Sergio R Filipe, Herminia De Lencastre, Alexander Tomasz
    Abstract:

    The essential function of Penicillin-Binding Protein 2 (PBP2) in methicillin-susceptible Staphylococcus aureus RN4220 was clearly established by placing the pbp2 gene under control of the inducible Pspac promoter; the resulting bacteria were unable to grow in the absence of inducer. In contrast, the deficit in PBP2 caused by inhibition of transcription of the pbp2 gene did not block growth of a methicillin-resistant S. aureus strain expressing the extra Penicillin-Binding Protein PBP2A, a Protein of extraspecies origin that is central to the mechanism of methicillin resistance. Several lines of evidence indicate that the essential function of PBP2 that can be compensated for by PBP2A is the transpeptidase activity. This provides direct genetic evidence that PBP2A has transpeptidase activity.

André Zapun - One of the best experts on this subject based on the ideXlab platform.

  • Inhibition of Streptococcus pneumoniae Penicillin-Binding Protein 2x and Actinomadura R39 DD-peptidase activities by ceftaroline.
    Antimicrobial Agents and Chemotherapy, 2013
    Co-Authors: Astrid Zervosen, André Zapun, Jean-marie Frère
    Abstract:

    Although the rate of acylation of a Penicillin-resistant form of Streptococcus pneumoniae Penicillin-Binding Protein 2x (PBP2x) by ceftaroline is 80-fold lower than that of its Penicillin-sensitive counterpart, it remains sufficiently high (k(2)/K = 12,600 M(-1) s(-1)) to explain the sensitivity of the Penicillin-resistant strain to this new cephalosporin. Surprisingly, the Actinomadura R39 DD-peptidase is not very sensitive to ceftaroline.

  • the membrane anchor of Penicillin Binding Protein pbp2a from streptococcus pneumoniae influences peptidoglycan chain length
    FEBS Journal, 2012
    Co-Authors: Waldemar Vollmer, André Zapun, Nordine Helassa, Eefjan Breukink, Thierry Vernet
    Abstract:

    The pneumococcus is an important Gram-positive pathogen, which shows increasing resistance to antibiotics, including β-lactams that target peptidoglycan assembly. Understanding cell-wall synthesis, at the molecular and cellular level, is essential for the prospect of combating drug resistance. As a first step towards reconstituting pneumococcal cell-wall assembly in vitro, we present the characterization of the glycosyltransferase activity of Penicillin-Binding Protein (PBP)2a from Streptococcus pneumoniae. Recombinant full-length membrane-anchored PBP2a was purified by ion-exchange chromatography. The glycosyltransferase activity of this enzyme was found to differ from that of a truncated periplasmic form. The full-length Protein with its cytoplasmic and transmembrane segment synthesizes longer glycan chains than the shorter form. The transpeptidase active site was functional, as shown by its reactivity towards bocillin and the catalysis of the hydrolysis of a thiol-ester substrate analogue. However, PBP2a did not cross-link the peptide stems of glycan chains in vitro. The absence of transpeptidase activity indicates that an essential component is missing from the in vitro system.

Johannes G Kusters - One of the best experts on this subject based on the ideXlab platform.

  • multiple mutations in or adjacent to the conserved Penicillin Binding Protein motifs of the Penicillin Binding Protein 1a confer amoxicillin resistance to helicobacter pylori
    Helicobacter, 2006
    Co-Authors: M M Gerrits, Ernst J Kuipers, Anita P O Godoy, Marcelo Lima Ribeiro, Jeroen Stoof, Sergio Mendonca, Arnoud H M Van Vliet, Jose Pedrazzoli, Johannes G Kusters
    Abstract:

    Background:  Amoxicillin-based therapies are highly effective for the treatment of Helicobacter pylori infections, but the efficacy may decrease as the incidence of amoxicillin resistance is increasing. So far, the molecular mechanism underlying stable amoxicillin resistance has only been identified for a few naturally occurring amoxicillin-resistant (AmxR) H. pylori isolates, and is mediated by mutations in Penicillin-Binding Protein 1A (PBP1A). In this study the molecular mechanism underlying amoxicillin resistance of seven additional AmxR H. pylori isolates has been established. Methods: H. pylori strain 26695 (minimal inhibitory concentration (MIC) 0.125 mg/l) was naturally transformed with total DNA and pbp1A polymerase chain reaction (PCR) products from the seven AmxR H. pylori isolates, and the MIC of amoxicillin and pbp1A gene sequence of the obtained AmxR transformants were determined. Results:  Replacement of the wild-type pbp1A gene of H. pylori reference strain 26695 by the pbp1A gene of the AmxR H. pylori isolates resulted in an increased MIC (0.5–1.0 mg/l). Sequence analysis of the smallest PBP1A fragments able to transfer the resistance indicated that several amino acid substitutions in or adjacent to the second (SKN402−404) and third (KTG555−557) conserved Penicillin-Binding Protein motifs (PBP-motifs) mediate amoxicillin resistance in H. pylori. This was confirmed by site-directed mutagenesis using oligonucleotides that contained defined mutations in or adjacent to these PBP-motifs. Conclusion:  In naturally occurring AmxR H. pylori isolates, amoxicillin resistance is mediated by various mutational changes located in or adjacent to the second and third PBP-motifs of the PBP1A. Although we cannot exclude the role of the other genes in amoxicillin resistance, it is likely that multiple mutational changes in the PBP1A gene are the predominant cause of amoxicillin resistance in H. pylori. The findings of this study currently preclude the rapid detection of amoxicillin resistance in H. pylori by molecular tests.

  • alterations in Penicillin Binding Protein 1a confer resistance to β lactam antibiotics in helicobacter pylori
    Antimicrobial Agents and Chemotherapy, 2002
    Co-Authors: M M Gerrits, D Schuijffel, A A Van Zwet, Ernst J Kuipers, Christina M J E Vandenbrouckegrauls, Johannes G Kusters
    Abstract:

    Most Helicobacter pylori strains are susceptible to amoxicillin, an important component of combination therapies for H. pylori eradication. The isolation and initial characterization of the first reported stable amoxicillin-resistant clinical H. pylori isolate (the Hardenberg strain) have been published previously, but the underlying resistance mechanism was not described. Here we present evidence that the β-lactam resistance of the Hardenberg strain results from a single amino acid substitution in HP0597, a Penicillin-Binding Protein 1A (PBP1A) homolog of Escherichia coli. Replacement of the wild-type HP0597 (pbp1A) gene of the amoxicillin-sensitive (Amxs) H. pylori strain 1061 by the Hardenberg pbp1A gene resulted in a 100-fold increase in the MIC of amoxicillin. Sequence analysis of pbp1A of the Hardenberg strain, the Amxs H. pylori strain 1061, and four amoxicillin-resistant (Amxr) 1061 transformants revealed a few amino acid substitutions, of which only a single Ser414→Arg substitution was involved in amoxicillin resistance. Although we cannot exclude that mutations in other genes are required for high-level amoxicillin resistance of the Hardenberg strain, this amino acid substitution in PBP1A resulted in an increased MIC of amoxicillin that was almost identical to that for the original Hardenberg strain.

Bum Han Ryu - One of the best experts on this subject based on the ideXlab platform.

  • Identification and Crystallization of Penicillin-Binding Protein/β-Lactamase Homolog (Rp46) from Ruegeria Pomeroyi
    Crystals, 2016
    Co-Authors: Bum Han Ryu, Tri Duc Ngo, Wanki Yoo, Kyeong Kyu Kim, T. Doohun Kim
    Abstract:

    In spite of the enormous biological and clinical significance of Penicillin-Binding Protein (PBP)/β-lactamase (βL), few of their many homologs (PBP)/βLs homologs) have been studied crystallographically, and have known functions. Herein, X-ray crystallographic study of a PBP/βL homolog (Rp46) from Ruegeria pomeroyi is described. Multiple sequence alignments indicate that Rp46 has a conserved serine residue within the S70-X-X-K73 motif (Motif I), acting as the catalytic nucleophile. Moreover, an invariant tyrosine residue (Tyr185) and a Trp365-X-Gly motif (Motif III) were also identified. The recombinant Rp46 Protein was expressed in Escherichia coli and purified to homogeneity judging from the SDS-PAGE analysis. Rp46 was crystallized using a solution consisting of 20% (w/v) PEG 3000, 0.1 M Tris-HCl, pH 7.0, 0.2 M calcium acetate, and the X-ray diffraction data were collected to a resolution of 1.90 Å with an Rmerge of 7.4%. The crystals of Rp46 belong to the space group I422, with unit cell parameters a = b = 141.26 Å, and c = 119.75. The structure determination and biochemical characterization are in progress. (Synopsis: A Penicillin-Binding Protein/β-lactamase homolog (Rp46) from Ruegeria pomeroyi was identified and crystallized in the space group I4, and the diffraction data were collected to a resolution of 1.90 Å.

  • identification and crystallization of Penicillin Binding Protein β lactamase homolog rp46 from ruegeria pomeroyi
    Crystals, 2016
    Co-Authors: Bum Han Ryu, Tri Duc Ngo, Wanki Yoo, Kyeong Kyu Kim, Doohun T Kim
    Abstract:

    In spite of the enormous biological and clinical significance of Penicillin-Binding Protein (PBP)/β-lactamase (βL), few of their many homologs (PBP)/βLs homologs) have been studied crystallographically, and have known functions. Herein, X-ray crystallographic study of a PBP/βL homolog (Rp46) from Ruegeria pomeroyi is described. Multiple sequence alignments indicate that Rp46 has a conserved serine residue within the S70-X-X-K73 motif (Motif I), acting as the catalytic nucleophile. Moreover, an invariant tyrosine residue (Tyr185) and a Trp365-X-Gly motif (Motif III) were also identified. The recombinant Rp46 Protein was expressed in Escherichia coli and purified to homogeneity judging from the SDS-PAGE analysis. Rp46 was crystallized using a solution consisting of 20% (w/v) PEG 3000, 0.1 M Tris-HCl, pH 7.0, 0.2 M calcium acetate, and the X-ray diffraction data were collected to a resolution of 1.90 A with an Rmerge of 7.4%. The crystals of Rp46 belong to the space group I422, with unit cell parameters a = b = 141.26 A, and c = 119.75. The structure determination and biochemical characterization are in progress. (Synopsis: A Penicillin-Binding Protein/β-lactamase homolog (Rp46) from Ruegeria pomeroyi was identified and crystallized in the space group I4, and the diffraction data were collected to a resolution of 1.90 A.)