Slow Binding Inhibition of Phospho-N-acetylmuramyl-pentapeptide-translocase (Escherichia coli) by Mureidomycin A

Brandish, Philip E.; Burnham, Martin K. R.; Lonsdale, John T.; Southgate, Robert; Inukai, Masatoshi; Bugg, Timothy D. H.

doi:10.1074/jbc.271.13.7609

Cited by 105 publications

(151 citation statements)

References 30 publications

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“…6A, MraY activity is dramatically reduced in membranes isolated after Emyc expression relative to the activity in control membranes. This level of inhibition was similar to that caused by the MraY inhibitor tunicamycin (25).…”

Section: E Is a Cell Wall Synthesissupporting

confidence: 73%

“…Rfe catalyzes the formation of the first lipid intermediate in enterobacterial common antigen synthesis, undecaprenol-P-P-GlcNAc (26). To determine whether Emyc is a specific inhibitor of MraY like the antibiotic mureidomycin (27) or a general GPT enzyme inhibitor like tunicamycin (25,26), we measured Rfe activity in the membranes described above using an exchange reaction similar to that catalyzed by MraY. Rfe activity was nearly identical in membranes isolated with and without prior Emyc expression (Fig.…”

Section: E Is a Cell Wall Synthesismentioning

confidence: 99%

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The Lysis Protein E of φX174 Is a Specific Inhibitor of the MraY-catalyzed Step in Peptidoglycan Synthesis

Bernhardt

Struck

Young

2001

Journal of Biological Chemistry

107

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Coliphage X174 encodes a single lysis protein, E, a 91-amino acid membrane protein. Dominant mutations have been isolated in the host gene mraY that confer E resistance. mraY encodes translocase I, which catalyzes the formation of the first lipid intermediate in bacterial cell wall synthesis, suggesting a model in which E inhibits MraY and promotes cell lysis in a manner analogous to cell wall synthesis inhibitors like penicillin. To test this model biochemically, we monitored the effect of E on cell wall synthesis in vivo and in vitro. We find that expression of Emyc, encoding an epitope-tagged E protein, from a multicopy plasmid inhibits the incorporation of [ 3 H]diaminopimelic acid into cell wall and leads to a profile of labeled precursors consistent with MraY inhibition. Moreover, we find that membranes isolated after Emyc expression are drastically reduced in MraY activity, whereas the activity of Rfe, an enzyme in the same superfamily, was unaffected. We therefore conclude that E is indeed a cell wall synthesis inhibitor and that this inhibition results from a specific block at the MraY-catalyzed step in the pathway.There are at least two distinct mechanisms by which phage promote destruction of the bacterial cell wall and subsequent cell lysis, the choice of which appears to be determined by the phage genome size. Bacteriophages with large genomes encode a holin-endolysin system. In the prototypic model, the S holin protein accumulates in the cell membrane and the R endolysin accumulates in the cytoplasm. At a genetically programmed time, S forms a membrane lesion to release the R endolysin into the periplasm where it can degrade the cell wall and cause lysis (1). In contrast, bacteriophages with small genomes can only afford to encode a single lysis protein. Three unrelated single protein lysis systems are known: the E protein from X174 (ssDNA, Microviridae), and the L and A 2 proteins from MS2 (ssRNA, group I) and Q␤ (ssRNA, group III) respectively (1-5). The molecular mechanism of lysis caused by any of these proteins has been elusive. No cell wall-degrading activity has been associated with any of these phages, indicating that their lytic mechanism is distinct from the holin-endolysin system of the larger phages.Working from the premise that these phage proteins are targeting a host protein to promote lysis, we took a genetic approach to isolate lysis-resistant host mutants and identify the target genes. We recently isolated dominant mutations in Escherichia coli mraY that result in resistance to the bacteriophage X174 E lysis protein (6). mraY encodes translocase I, which catalyzes the formation of the first lipid intermediate in cell wall synthesis (7) and is a member of the UDP-GlcNAc/ MurNAc 1 :polyisoprenyl-P GlcNAc/MurNAc 1-P transferase family of enzymes, which we will refer to as the GPT enzyme family (8). Inhibition of MraY by the antibiotic mureidomycin results in lysis of Pseudomonas aeruginosa, and depletion of MraY activity from E. coli also results in concomitant cell lysis (9, 10). Th...

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Section: E Is a Cell Wall Synthesissupporting

confidence: 73%

Section: E Is a Cell Wall Synthesismentioning

confidence: 99%

The Lysis Protein E of φX174 Is a Specific Inhibitor of the MraY-catalyzed Step in Peptidoglycan Synthesis

Bernhardt

Struck

Young

2001

Journal of Biological Chemistry

107

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“…Fluorescence-based determination of MurF activity Brandish et al 31 reported the assay to measure translocase I activity by determining the difference in fluorescent intensities of the substrate UDP-MurNAc-dansylpentapeptide and the product dansylated lipid I. We applied this method to high-throughput screening of natural products and discovered novel translocase I inhibitors.…”

Section: Resultsmentioning

confidence: 99%

A novel assay of bacterial peptidoglycan synthesis for natural product screening

et al. 2009

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Although a large number of microbial metabolites have been discovered as inhibitors of bacterial peptidoglycan biosynthesis, only a few inhibitors were reported for the pathway of UDP-MurNAc-pentapeptide formation, partly because of the lack of assays appropriate for natural product screening. Among the pathway enzymes, D-Ala racemase (Alr), D-Ala:D-Ala ligase (Ddl) and UDPMurNAc-tripeptide:D-Ala-D-Ala transferase (MurF) are particularly attractive as antibacterial targets, because these enzymes are essential for growth and utilize low-molecular-weight substrates. Using dansylated UDP-MurNAc-tripeptide and L-Ala as the substrates, we established a cell-free assay to measure the sequential reactions of Alr, Ddl and MurF coupled with translocase I. This assay is sensitive and robust enough to screen mixtures of microbial metabolites, and enables us to distinguish the inhibitors for D-Ala-D-Ala formation, MurF and translocase I. D-cycloserine, the D-Ala-D-Ala pathway inhibitor, was successfully detected by this assay (IC 50 ¼1.7 lg ml À1 ). In a large-scale screening of natural products, we have identified inhibitors for D-Ala-D-Ala synthesis pathway, MurF and translocase I.

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“…The biosynthetic pathway to both the pacidamycin [36][37][38] and caprazamycin [39] antibiotics has been elucidated, hence the use of biosynthetic engineering offers the possibility of generating modified uridyl peptide antibiotics via fermentation in the future. shown that mureidomycin A and liposidomycin B are both slow-binding inhibitors, while tunicamycin is a reversible inhibitor [40,41]. Inhibition by mureidomycin A was found to be competitive with both UDPMurNAc-pentapeptide and undecaprenyl phosphate substrates, whereas liposidomycin B was competitive with undecaprenyl phosphate, but non-competitive with UDPMurNAc-pentapeptide [41].…”

Section: Nucleoside Natural Product Inhibitors Of Mray and Related Enmentioning

confidence: 96%

“…shown that mureidomycin A and liposidomycin B are both slow-binding inhibitors, while tunicamycin is a reversible inhibitor [40,41]. Inhibition by mureidomycin A was found to be competitive with both UDPMurNAc-pentapeptide and undecaprenyl phosphate substrates, whereas liposidomycin B was competitive with undecaprenyl phosphate, but non-competitive with UDPMurNAc-pentapeptide [41].…”

Section: Nucleoside Natural Product Inhibitors Of Mray and Related Enmentioning

confidence: 96%

Inhibition of phospho-MurNAc-pentapeptide translocase (MraY) by nucleoside natural product antibiotics, bacteriophage ϕX174 lysis protein E, and cationic antibacterial peptides

Bugg

Rodolis

Mihalyi

et al. 2016

Bioorganic & Medicinal Chemistry

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Slow Binding Inhibition of Phospho-N-acetylmuramyl-pentapeptide-translocase (Escherichia coli) by Mureidomycin A

Cited by 105 publications

References 30 publications

The Lysis Protein E of φX174 Is a Specific Inhibitor of the MraY-catalyzed Step in Peptidoglycan Synthesis

The Lysis Protein E of φX174 Is a Specific Inhibitor of the MraY-catalyzed Step in Peptidoglycan Synthesis

A novel assay of bacterial peptidoglycan synthesis for natural product screening

Inhibition of phospho-MurNAc-pentapeptide translocase (MraY) by nucleoside natural product antibiotics, bacteriophage ϕX174 lysis protein E, and cationic antibacterial peptides

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