Identification and Characterization of a Monofunctional Glycosyltransferase from
            <i>Staphylococcus aureus</i>

Wang, Q. May; Peery, Robert B.; Johnson, Robert B.; Alborn, William E.; Yeh, Wu-Kuang; Skatrud, Paul L.

doi:10.1128/jb.183.16.4779-4785.2001

Cited by 78 publications

(67 citation statements)

References 24 publications

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“…The membrane association sites in E. coli PBP1b and in S. pneumoniae PBP2a* encompass only the first GT motif, whereas in Mycobacterium leprae PBP1, the region comprising the third, fourth, and fifth GT motifs contains the secondary membrane binding site (4,6,21,36). Monofunctional GT proteins from Staphylococcus aureus and Ralstonia eutropha deprived from their transmembrane anchors were also shown to aggregate (26,37). This putative membrane association of the GT domain from class A PBPs is consistent with the membrane localization of the substrate lipid II.…”

Section: Discussionmentioning

confidence: 99%

Functional Characterization of Penicillin-Binding Protein 1b fromStreptococcus pneumoniae

et al. 2003

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The widespread use of antibiotics has encouraged the development of drug resistance in pathogenic bacteria. In order to overcome this problem, the modification of existing antibiotics and/or the identification of targets for the design of new antibiotics is currently being undertaken. Bifunctional penicillin-binding proteins (PBPs) are membrane-associated molecules whose transpeptidase (TP) activity is irreversibly inhibited by ␤-lactam antibiotics and whose glycosyltransferase (GT) activity represents a potential target in the antibacterial fight. In this work, we describe the expression and the biochemical characterization of the soluble extracellular region of Streptococcus pneumoniae PBP1b (PBP1b*). The acylation efficiency for benzylpenicillin and cefotaxime was characterized by stopped-flow fluorometry and a 40-kDa stable TP domain was generated after limited proteolysis. In order to analyze the GT activity of PBP1b*, we developed an electrophoretic assay which monitors the fluorescence signal from PBP1b*-bound dansylated lipid II. This binding was inhibited by the antibiotic moenomycin and was specific for the GT domain, since no signal was observed in the presence of the purified functional TP domain. Binding studies performed with truncated forms of PBP1b* demonstrated that the first conserved motif of the GT domain is not required for the recognition of lipid II, whereas the second motif is necessary for such interaction.

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Section: Discussionmentioning

confidence: 99%

Functional Characterization of Penicillin-Binding Protein 1b fromStreptococcus pneumoniae

et al. 2003

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“…In Staphylococcus aureus, expression of methicillin resistance requires the glycosyltransferase activity of the sole class A PBP produced by this species (Pbp2) and the D,D-transpeptidase activity of a low-affinity class B enzyme (Pbp2a) (10). However, neither activity is required for growth in the absence of antibiotics since polymerization of glycan chains can alternatively be performed by a monofunctional glycosyltransferase (Mgt), a representative of an enzyme family which displays sequence similarity to the glycosyltransferase domain of class A PBPs (17). These phenotypes imply the existence of a functional interaction between the D,D-transpeptidase domain of Pbp2a and the glycosyltransferase domain of PBP2, which is essential for peptidoglycan synthesis (10,17).…”

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Role of Class A Penicillin-Binding Proteins in the Expression of β-Lactam Resistance in Enterococcus faecium

et al. 2009

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Peptidoglycan is polymerized by monofunctional D,D-transpeptidases belonging to class B penicillin-binding proteins (PBPs) and monofunctional glycosyltransferases and by bifunctional enzymes that combine both activities (class A PBPs). Three genes encoding putative class A PBPs (pbpF, pbpZ, and ponA) were deleted from the chromosome of Enterococcus faecium D344R in all possible combinations in order to identify the glycosyltransferases that cooperate with low-affinity class B Pbp5 for synthesis of peptidoglycan in the presence of ␤-lactam antibiotics. The viability of the triple mutant indicated that glycan strands can be polymerized independently from class A PBPs by an unknown glycosyltranferase. The susceptibility of the ⌬pbpF ⌬ponA mutant and triple mutants to extended spectrum cephalosporins (ceftriaxone and cefepime) identified either PbpF or PonA as essential partners of Pbp5 for peptidoglycan polymerization in the presence of the drugs. Mass spectrometry analysis of peptidoglycan structure showed that loss of PonA and PbpF activity led to a minor decrease in the extent of peptidoglycan cross-linking by the remaining PBPs without any detectable compensatory increase in the participation of the L,D-transpeptidase in peptidoglycan synthesis. Optical density measurements and electron microscopy analyses showed that the ⌬pbpF ⌬ponA mutant underwent increased stationary-phase autolysis compared to the parental strain. Unexpectedly, deletion of the class A pbp genes revealed dissociation between the expression of resistance to cephalosporins and penicillins, although the production of Pbp5 was required for resistance to both classes of drugs. Thus, susceptibility of Pbp5-mediated peptidoglycan cross-linking to different ␤-lactam antibiotics differed as a function of its partner glycosyltransferase.

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“…Both classes have C-terminal penicillin-binding domains containing the transpeptidase activity that cross-links peptide side chains. Some species also have monofunctional glycosyltransferases (MGTs) that contain the conserved amino acid sequences found in the class A PBP GT domain but which lack a penicillin-binding domain (7,39 The fact that many species contain multiple class A PBPs (10) and that removal of one or more of those proteins results in little or no effect on PG polymerization or cell viability demonstrates their functional redundancies (6,24,33). However, a function of class A PBPs has been demonstrated to be essential in both Escherichia coli and Streptococcus pneumoniae.…”

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Peptidoglycan Synthesis in the Absence of Class A Penicillin-Binding Proteins in Bacillus subtilis

2003

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Penicillin-binding proteins (PBPs) catalyze the final, essential reactions of peptidoglycan synthesis. Three classes of PBPs catalyze either trans-, endo-, or carboxypeptidase activities on the peptidoglycan peptide side chains. Only the class A high-molecular-weight PBPs have clearly demonstrated glycosyltransferase activities that polymerize the glycan strands, and in some species these proteins have been shown to be essential. The Bacillus subtilis genome sequence contains four genes encoding class A PBPs and no other genes with similarity to their glycosyltransferase domain. A strain lacking all four class A PBPs has been constructed and produces a peptidoglycan wall with only small structural differences from that of the wild type. The growth rate of the quadruple mutant is much lower than those of strains lacking only three of the class A PBPs, and increases in cell length and frequencies of wall abnormalities were noticeable. The viability and wall production of the quadruple-mutant strain indicate that a novel enzyme can perform the glycosyltransferase activity required for peptidoglycan synthesis. This activity was demonstrated in vitro and shown to be sensitive to the glycosyltransferase inhibitor moenomycin. In contrast, the quadruple-mutant strain was resistant to moenomycin in vivo. Exposure of the wild-type strain to moenomycin resulted in production of a phenotype similar to that of the quadruple mutant.The structural element of the bacterial cell wall is peptidoglycan (PG), and the importance of this structure is apparent in the number of antimicrobial agents targeting its components and the enzymatic reactions leading to its synthesis. The glycosyltransferase (GT) activity carrying out one of the final enzymatic reactions in PG synthesis is an attractive target, because the enzymes performing that activity are highly conserved and believed to be essential (9, 10). PG is composed of glycan strands cross-linked by peptide side chains. The glycan strands are synthesized by a GT that adds lipid-linked disaccharide pentapeptide subunits to nascent glycan strands. The pentapeptides are then utilized by a transpeptidase to crosslink adjacent glycan strands (reviewed in reference 4). Most of the proteins involved in these final enzymatic reactions are penicillin-binding proteins (PBPs), which are divided into three classes based on the presence of conserved functional domains (9, 10). Only the class A PBPs have an N-terminal domain that contains the conserved amino acid sequences found in all GTs clearly demonstrated to polymerize PG. Class B PBPs have a different N-terminal domain; and although some researchers have associated GT activity with class B PBPs (15), others have been unable to reproduce those results (2). Both classes have C-terminal penicillin-binding domains containing the transpeptidase activity that cross-links peptide side chains. Some species also have monofunctional glycosyltransferases (MGTs) that contain the conserved amino acid sequences found in the class A PBP GT domain but whic...

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Identification and Characterization of a Monofunctional Glycosyltransferase from Staphylococcus aureus

Cited by 78 publications

References 24 publications

Functional Characterization of Penicillin-Binding Protein 1b fromStreptococcus pneumoniae

Functional Characterization of Penicillin-Binding Protein 1b fromStreptococcus pneumoniae

Role of Class A Penicillin-Binding Proteins in the Expression of β-Lactam Resistance in Enterococcus faecium

Peptidoglycan Synthesis in the Absence of Class A Penicillin-Binding Proteins in Bacillus subtilis

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