Assay for Identification of Inhibitors for Bacterial MraY Translocase or MurG Transferase

Branstrom, Arthur; Midha, Sunita; Longley, Clifford; Han, Kiho; Baizman, Eugene R.; Axelrod, Helena R.

doi:10.1006/abio.2000.4530

Cited by 37 publications

(43 citation statements)

References 11 publications

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“…Lipid products were purified by extraction with 100 l of n-butanol and quantitated by liquid scintillation counting. Control experiments omitting undecaprenyl phosphate showed that rates of accumulation of n-butanol-soluble 14 C were 0 to 12% of those obtained in the presence of undecaprenyl phosphate. Control experiments performed in the presence of mureidomycin A (10 M) (13,28,29) resulted in a complete loss of undecaprenyl phosphate-dependent incorporation of 14 C into the n-butanol phase.…”

Section: Chemicalsmentioning

confidence: 91%

“…Mouse monoclonal anti-His 6 antibody and Pwo DNA polymerase were from Roche, goat anti-mouse antibody conjugated to HRP was from Promega, and rabbit anti-maltose binding protein (anti-MBP) antibody and goat anti-rabbit antibody were from New England Biolabs, Hitchin, United Kingdom. Protein assay reagents were from Pierce Inc. UDP-MurNAc-L-Ala-␥-D-Glu-m-DAP- 14 C-D-Ala-14 C-D-Ala was a gift of SmithKline Beecham Pharmaceuticals. UDP-MurNAc-pentapeptide was prepared as previously described (38).…”

Section: Chemicalsmentioning

confidence: 99%

“…Gene knockout experiments have demonstrated conclusively that the mraY gene is essential to cell viability in both E. coli and the gram-positive pathogen Streptococcus pneumoniae and is therefore an attractive target for antimicrobial drug development (6,11,14,48). Several nucleoside natural product antibiotics that contain the uridine moiety of the natural substrate of translocase 1 have been shown to be potent inhibitors of this enzyme, namely, the mureidomycins, the liposidomycins, tunicamycin, and, most recently, the muramycins (12,13,28,29,32,37).…”

mentioning

confidence: 99%

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Phospho- N -Acetyl-Muramyl-Pentapeptide Translocase from Escherichia coli : Catalytic Role of Conserved Aspartic Acid Residues

et al. 2004

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Phospho-N-acetyl-muramyl-pentapeptide translocase (translocase 1) catalyzes the first of a sequence of lipid-linked steps that ultimately assemble the peptidoglycan layer of the bacterial cell wall. This essential enzyme is the target of several natural product antibiotics and has recently been the focus of antimicrobial drug discovery programs. The catalytic mechanism of translocase 1 is believed to proceed via a covalent intermediate formed between phospho-N-acetyl-muramyl-pentapeptide and a nucleophilic amino acid residue. Amino acid sequence alignments of the translocase 1 family and members of the related transmembrane phosphosugar transferase superfamily revealed only three conserved residues that possess nucleophilic side chains: the aspartic acid residues D115, D116, and D267. Here we report the expression and partial purification of Escherichia coli translocase 1 as a C-terminal hexahistidine (C-His 6 ) fusion protein. Three enzymes with the site-directed mutations D115N, D116N, and D267N were constructed, expressed, and purified as C-His 6 fusions. Enzymatic analysis established that all three mutations eliminated translocase 1 activity, and this finding verified the essential role of these residues. By analogy with the structural environment of the double aspartate motif found in prenyl transferases, we propose a model whereby D115 and D116 chelate a magnesium ion that coordinates with the pyrophosphate bridge of the UDP-N-acetyl-muramyl-pentapeptide substrate and in which D267 therefore fulfills the role of the translocase 1 active-site nucleophile.Enzymes involved in the assembly of the peptidoglycan layer of bacterial cell walls represent important targets for antibacterial chemotherapy (15, 49). The study of this class of enzymes and the search for selective inhibitors are likely to lead to the development of new chemotherapeutic agents, which are urgently needed to combat antimicrobial drug resistance, the threat of which has recently been highlighted by the acquisition of resistance by methicillin-resistant Staphylococcus aureus to vancomycin (43).Peptidoglycan consists of a ␤-1,4-linked N-acetyl-glucosamine-N-acetyl-muramyl-pentapeptide (GlcNAc-MurNAc-pentapeptide) polymer, assembled from cytoplasmic precursors UDP-MurNAc-L-Ala-␥-D-Glu-X-D-Ala-D-Ala (UDPMurNAc-pentapeptide; X, L-Lys or meso-diaminopimelic acid [meso-DAP]) and 49

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Section: Chemicalsmentioning

confidence: 91%

Section: Chemicalsmentioning

confidence: 99%

mentioning

confidence: 99%

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Phospho- N -Acetyl-Muramyl-Pentapeptide Translocase from Escherichia coli : Catalytic Role of Conserved Aspartic Acid Residues

et al. 2004

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“…Over the past few years, new enzymatic assays mostly designed for high-throughput screening and the study of specific inhibitors were developed VOL. 71,2007 PEPTIDOGLYCAN LIPID INTERMEDIATES 623 (26,92,159,161,200). As a target in the search for novel antibacterials, MraY presents many advantages (essentiality, ubiquity to eubacteria, specificity).…”

Section: Biosynthesis Of Lipid Imentioning

confidence: 99%

“…Owing to its difficult availability in large amounts (136,166), assays based on a reaction coupled with MraY were developed (45,121,160). Various coupled assays have since been used for high-throughput screening purposes (26,50,92,140,200). Direct assays with synthetic lipid I analogues were first carried out with compounds containing C 10 and C 35 chains (10,11,119).…”

Section: Biosynthesis Of Lipid IImentioning

confidence: 99%

Lipid Intermediates in the Biosynthesis of Bacterial Peptidoglycan

Heijenoort

2007

Microbiol Mol Biol Rev

172

135

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SUMMARY This review is an attempt to bring together and critically evaluate the now-abundant but dispersed data concerning the lipid intermediates of the biosynthesis of bacterial peptidoglycan. Lipid I, lipid II, and their modified forms play a key role not only as the specific link between the intracellular synthesis of the peptidoglycan monomer unit and the extracytoplasmic polymerization reactions but also in the attachment of proteins to the bacterial cell wall and in the mechanisms of action of antibiotics with which they form specific complexes. The survey deals first with their detection, purification, structure, and preparation by chemical and enzymatic methods. The recent important advances in the study of transferases MraY and MurG, responsible for the formation of lipids I and II, are reported. Various modifications undergone by lipids I and II are described, especially those occurring in gram-positive organisms. The following section concerns the cellular location of the lipid intermediates and the translocation of lipid II across the cytoplasmic membrane. The great efforts made since 2000 in the study of the glycosyltransferases catalyzing the glycan chain formation with lipid II or analogues are analyzed in detail. Finally, examples of antibiotics forming complexes with the lipid intermediates are presented.

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A Multitarget Assay for Inhibitors of Membrane-Associated Steps of Peptidoglycan Biosynthesis

Barbosa

Ross

Hillman

et al. 2002

Analytical Biochemistry

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Assay for Identification of Inhibitors for Bacterial MraY Translocase or MurG Transferase

Cited by 37 publications

References 11 publications

Phospho- N -Acetyl-Muramyl-Pentapeptide Translocase from Escherichia coli : Catalytic Role of Conserved Aspartic Acid Residues

Phospho- N -Acetyl-Muramyl-Pentapeptide Translocase from Escherichia coli : Catalytic Role of Conserved Aspartic Acid Residues

Lipid Intermediates in the Biosynthesis of Bacterial Peptidoglycan

A Multitarget Assay for Inhibitors of Membrane-Associated Steps of Peptidoglycan Biosynthesis

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