The University of Southampton
University of Southampton Institutional Repository

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
Phospho-N-acetyl-muramyl-pentapeptide translocase from Escherichia coli: catalytic role of conserved aspartic acid residues
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, fusions. Enzymatic analysis established that all three mutations eliminated translocase I 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.
farnesyl diphosphate synthase, peptidoglycan synthesis, mureidomycin-a, polyacrylamide gels, initial stage, mray, proteins, biosynthesis, transferase, membrane
0021-9193
1747-1757
Lloyd, Adrian J.
f91c1291-d23b-4436-bcc5-255c908a3527
Brandish, Phillip H.
cd737599-d640-4774-afce-920025aea045
Gilbey, Andrea M.
74fae8ef-5eec-440a-9a64-6bc5988bcf49
Bugg, Timothy D.H.
300f9ac5-0185-438a-a930-9bf22c08ddd5
Lloyd, Adrian J.
f91c1291-d23b-4436-bcc5-255c908a3527
Brandish, Phillip H.
cd737599-d640-4774-afce-920025aea045
Gilbey, Andrea M.
74fae8ef-5eec-440a-9a64-6bc5988bcf49
Bugg, Timothy D.H.
300f9ac5-0185-438a-a930-9bf22c08ddd5

Lloyd, Adrian J., Brandish, Phillip H., 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), 1747-1757. (doi:10.1128/JB.186.6.1747-1757.2004).

Record type: Article

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-His(6)) fusion protein. Three enzymes with the site-directed mutations D115N, D116N, and D267N were constructed, expressed, and purified as C-His, fusions. Enzymatic analysis established that all three mutations eliminated translocase I 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.

This record has no associated files available for download.

More information

Published date: 1 March 2004
Keywords: farnesyl diphosphate synthase, peptidoglycan synthesis, mureidomycin-a, polyacrylamide gels, initial stage, mray, proteins, biosynthesis, transferase, membrane

Identifiers

Local EPrints ID: 20276
URI: http://eprints.soton.ac.uk/id/eprint/20276
ISSN: 0021-9193
PURE UUID: 1305e61c-2634-469e-bccf-aa3f1d1ac53f

Catalogue record

Date deposited: 20 Feb 2006
Last modified: 15 Mar 2024 06:23

Export record

Altmetrics

Contributors

Author: Adrian J. Lloyd
Author: Phillip H. Brandish
Author: Andrea M. Gilbey
Author: Timothy D.H. Bugg

Download statistics

Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.

View more statistics

Atom RSS 1.0 RSS 2.0

Contact ePrints Soton: eprints@soton.ac.uk

ePrints Soton supports OAI 2.0 with a base URL of http://eprints.soton.ac.uk/cgi/oai2

This repository has been built using EPrints software, developed at the University of Southampton, but available to everyone to use.

We use cookies to ensure that we give you the best experience on our website. If you continue without changing your settings, we will assume that you are happy to receive cookies on the University of Southampton website.

×