Journal of Molecular Biology
Regular articleThe structure of a LysM domain from E. coli membrane-bound lytic murein transglycosylase D (MltD)1☆,
Introduction
Peptidoglycan is a component of the cell walls of both Gram positive and Gram negative bacteria, where it provides mechanical support and prevents bacteria bursting under high internal osmotic pressure. It is composed of linear chains of N-acetylmuramic acid and N-acetylglucosamine, cross-linked by short peptides. The importance of peptidoglycan is highlighted by the fact that many antibiotics block its biosynthesis. Bacteria and bacteriophage produce a variety of enzymes that cleave peptidoglycan either to lyse host cells or to re-model the cell wall during growth or cell division. Different classes of peptidoglycan degrading enzymes cleave the molecule at different points. Some, such as muramidases and lysozymes, attack the glycosidic linkages of the glycan strand. Amidases cleave the amide bond between N-acetyl muramic acid and the l alanine that forms part of the peptide cross-links, and peptidases cut amide bonds in the peptide cross-links. Many of these enzymes are modular and are composed of catalytic units linked to a number of other domains. The extracellular muramidase 2 of Enterococcus hirae, for example, consists of a catalytic N-terminal domain fused to a C-terminal region containing multiple repeats of approximately 40 amino acid residues (Joris et al., 1992). This sequence motif is found in many other enzymes involved in cell wall degradation and is also present in a number of other proteins, many of which are known to be associated with bacterial cell walls (Birkeland, 1994)(Figure 1). It appears that this motif codes for a protein domain that probably has a general peptidoglycan binding function. Since it was originally identified in bacterial lysins, the repeats have been termed LysM domains for lysin motif (Ponting et al., 1999). To understand further the structure and function of this module we have determined the structure of a LysM domain from Escherichia coli membrane-bound lytic murein transglycosylase D (MltD). MltD consists of an N-terminal transglycosylase domain with two LysM repeats at the C terminus (Figure 1). The exact function of MltD is unknown. However, based on the phylogenetic distribution of the protein it has recently been suggested that it may be involved in flagellar biosynthesis (Pellegrini et al., 1999).
Section snippets
Results
Residues 389 to 452 of E. coli MltD encompassing the C-terminal copy of the two motifs in this enzyme, were overexpressed in E. coli and purified to homogeneity. Assignments for this peptide were obtained using a range of 2D and 3D heteronuclear NMR experiments using isotopically enriched samples. Non-random coil chemical shifts and long-range NOEs were only observed for the residues between 398 and 445, and structures were calculated for this region. Residues will now be referred to with
Discussion
Here, we have demonstrated that the lysin motif codes for a folded globular domain. A number of other domains have been characterised from bacterial cell surface proteins and it is becoming clear that these proteins, like many eukaryotic cell surface proteins, are highly modular in nature (Goward et al., 1993). A variety of enzymes involved in bacterial cell wall degradation, for example, contain a 60-amino acid residue domain distinct from the LysM domain that is also believed to have a
Sample preparation
The DNA sequence coding for residues 389 to 452 of E. coli MltD was amplified from E. coli chromosomal DNA using PCR and cloned into the pRSETa expression vector (Invitrogen). The LysM domain was expressed in C41 (DE3) cells (Miroux & Walker, 1996) grown in M9 media with appropriately labelled carbon and nitrogen sources. Protein was purified by ion exchange chromatography using DEAE fast flow Sepherose (Pharmacia) followed by gel filtration on a G75 Superdex column (Pharmacia). NMR samples
Supplementary Files
Acknowledgements
We thank Mark Proctor for expert sample preparation. This work was funded by the Wellcome Trust and the Medical Research Council.
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