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Phospho-N-acetyl-muramyl-pentapeptide translocase from Escherichia coli : catalytic role of conserved aspartic acid residues
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Lloyd, Adrian J., Brandish, Philip E., Gilbey, Andrea M. and Bugg, Timothy D. H. (2004) Phospho-N-acetyl-muramyl-pentapeptide translocase from Escherichia coli : catalytic role of conserved aspartic acid residues. Journal of bacteriology, 186 (6). pp. 1747-57. doi:10.1128/JB.186.6.1747-1757.2004 ISSN 0021-9193.
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Lloyd et al (2004) J. Bacteriol. 186 1747-57.pdf - Published Version Embargoed item. Restricted access to Repository staff only - Requires a PDF viewer. Download (720Kb) |
Official URL: https://doi.org/10.1128/JB.186.6.1747-1757.2004
Abstract
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-His6) fusion protein. Three enzymes with the site-directed mutations D115N, D116N, and D267N were constructed, expressed, and purified as C-His6 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.
| Item Type: | Journal Article | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) | ||||||||
| Journal or Publication Title: | Journal of bacteriology | ||||||||
| Publisher: | American Society for Microbiology | ||||||||
| ISSN: | 0021-9193 | ||||||||
| Official Date: | 15 March 2004 | ||||||||
| Dates: |
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| Volume: | 186 | ||||||||
| Number: | 6 | ||||||||
| Page Range: | pp. 1747-57 | ||||||||
| DOI: | 10.1128/JB.186.6.1747-1757.2004 | ||||||||
| Status: | Peer Reviewed | ||||||||
| Publication Status: | Published | ||||||||
| Access rights to Published version: | Restricted or Subscription Access | ||||||||
| Date of first compliant deposit: | 3 October 2022 | ||||||||
| Open Access Version: |
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