Purification and Characterization of the Bacterial MraY Translocase Catalyzing the First Membrane Step of Peptidoglycan Biosynthesis

Bouhss, Ahmed; Crouvoisier, Muriel; Blanot, Didier; Mengin‐Lecreulx, Dominique

doi:10.1074/jbc.m314165200

Cited by 154 publications

(208 citation statements)

References 36 publications

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“…A recent precedent is the inhibition of the transmembrane enzyme c-secretase by a-helical peptides at a transmembrane site removed from the active site (Das et al, 2003). MraY from Bacillus subtilis has recently been purified on a small scale (Bouhss et al, 2004); thus, it may be possible in future to carry out studies using homogeneous MraY protein. The availability of a biologically active synthetic peptide now allows more detailed studies of the E-MraY interaction.…”

Section: Discussionmentioning

confidence: 99%

Interaction of the transmembrane domain of lysis protein E from bacteriophage ϕX174 with bacterial translocase MraY and peptidyl-prolyl isomerase SlyD

et al. 2006

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The molecular target for the bacteriolytic E protein from bacteriophage wX174, responsible for host cell lysis, is known to be the enzyme phospho-MurNAc-pentapeptide translocase (MraY), an integral membrane protein involved in bacterial cell wall peptidoglycan biosynthesis, with an essential role being played by peptidyl-prolyl isomerase SlyD. A synthetic 37 aa peptide E pep , containing the N-terminal transmembrane a-helix of E, was found to be bacteriolytic against Bacillus licheniformis, and inhibited membrane-bound MraY. The solution conformation of E pep was found by circular dichroism (CD) spectroscopy to be 100 % a-helical. No change in the CD spectrum was observed upon addition of purified Escherichia coli SlyD, implying that SlyD does not catalyse prolyl isomerization upon E. However, E pep was found to be a potent inhibitor of SlyD-catalysed peptidylprolyl isomerization (IC 50 0?15 mM), implying a strong interaction between E and SlyD. E pep was found to inhibit E. coli MraY activity when assayed in membranes (IC 50 0?8 mM); however, no inhibition of solubilized MraY was observed, unlike nucleoside natural product inhibitor tunicamycin. These results imply that the interaction of E with MraY is not at the MraY active site, and suggest that a protein-protein interaction is formed between E and MraY at a site within the transmembrane region.

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Section: Discussionmentioning

confidence: 99%

Interaction of the transmembrane domain of lysis protein E from bacteriophage ϕX174 with bacterial translocase MraY and peptidyl-prolyl isomerase SlyD

et al. 2006

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“…E. coli mraY cloning followed the method previously described. 10 The mraY gene was cloned from chromosomal DNA of wild-type E. coli strain MG1655 using the polymerase chain reaction. Amplification was performed using High Fidelity PCR Master (Roche Applied Science, Indianapolis, IN) and the following primers (Eurofins MWG Operon, Huntsville, AL): 5′-AGGAACATGTCCCATCACCATCACCATCACAT-GTTAGTTTGGCTGGCCG-3′ and 5′-TAGGAGATCTTTA ACGTACCTTCAGCGTTGCC-3′.…”

Section: Methodsmentioning

confidence: 99%

A High-Throughput, Homogeneous, Fluorescence Resonance Energy Transfer-Based Assay for Phospho-N-acetylmuramoyl-pentapeptide Translocase (MraY)

et al. 2012

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Peptidoglycan biosynthesis is an essential process in bacteria and is therefore a suitable target for the discovery of new antibacterial drugs. One of the last cytoplasmic steps of peptidoglycan biosynthesis is catalyzed by the integral membrane protein MraY, which attaches soluble UDP-N-acetylmuramoyl-pentapeptide to the membrane-bound acceptor undecaprenyl phosphate. Although several natural product-derived inhibitors of MraY are known, none have the properties necessary to be of clinical use as antibacterial drugs. Here we describe a novel, homogeneous, fluorescence resonance energy transferbased MraY assay that is suitable for high-throughput screening for novel MraY inhibitors. The assay allows for continuous measurement, or it can be quenched prior to measurement.

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“…Once in the cytoplasm, the muropeptides are processed by various enzymes to form lipid II composed of UDPGlcNAcMurNac pentapeptide attached to a hydrophobic undecaprenol-pyrophosphate group [33,36]. Lipid II is flipped across the cytoplasmic membrane into the periplasmic space, where GlcNAcMurNac pentapeptide is reincorporated into the growing cell-wall (Figs 1 and 2).…”

Section: Cytoplasmmentioning

confidence: 99%

“…UDP-GlcNAc is further converted to UDP-MurNAc pentapeptide by the activity of the Mur group of enzymes (MurA, MurB, MurC, MurD, MurE and MurF) [31,32]. To this pentapeptide, a 55-carbon aliphatic chain called undecaprenyl pyrophosphate is attached by MraY [33]. This aliphatic moiety is attached to the inner face of the cytoplasmic membrane to form lipid I [34,35].…”

Section: Escherichia Colimentioning

confidence: 99%

Cell-wall recycling and synthesis in Escherichia coli and Pseudomonas aeruginosa – their role in the development of resistance

Dhar

Kumari

Balasubramanian

et al. 2018

Journal of Medical Microbiology

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The bacterial cell-wall that forms a protective layer over the inner membrane is called the murein sacculus -a tightly crosslinked peptidoglycan mesh unique to bacteria. Cell-wall synthesis and recycling are critical cellular processes essential for cell growth, elongation and division. Both de novo synthesis and recycling involve an array of enzymes across all cellular compartments, namely the outer membrane, periplasm, inner membrane and cytoplasm. Due to the exclusivity of peptidoglycan in the bacterial cell-wall, these players are the target of choice for many antibacterial agents. Our current understanding of cell-wall biochemistry and biogenesis in Gram-negative organisms stems mostly from studies of Escherichia coli. An incomplete knowledge on these processes exists for the opportunistic Gram-negative pathogen, Pseudomonas aeruginosa. In this review, cell-wall synthesis and recycling in the various cellular compartments are compared and contrasted between E. coli and P. aeruginosa. Despite the fact that there is a remarkable similarity of these processes between the two bacterial species, crucial differences alter their resistance to b-lactams, fluoroquinolones and aminoglycosides. One of the common mediators underlying resistance is the amp system whose mechanism of action is closely associated with the cell-wall recycling pathway. The activation of amp genes results in expression of AmpC blactamase through its cognate regulator AmpR which further regulates multi-drug resistance. In addition, other cell-wall recycling enzymes also contribute to antibiotic resistance. This comprehensive summary of the information should spawn new ideas on how to effectively target cell-wall processes to combat the growing resistance to existing antibiotics.

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Purification and Characterization of the Bacterial MraY Translocase Catalyzing the First Membrane Step of Peptidoglycan Biosynthesis

Cited by 154 publications

References 36 publications

Interaction of the transmembrane domain of lysis protein E from bacteriophage ϕX174 with bacterial translocase MraY and peptidyl-prolyl isomerase SlyD

Interaction of the transmembrane domain of lysis protein E from bacteriophage ϕX174 with bacterial translocase MraY and peptidyl-prolyl isomerase SlyD

A High-Throughput, Homogeneous, Fluorescence Resonance Energy Transfer-Based Assay for Phospho-N-acetylmuramoyl-pentapeptide Translocase (MraY)

Cell-wall recycling and synthesis in Escherichia coli and Pseudomonas aeruginosa – their role in the development of resistance

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