Journal of Molecular Biology
Regular articleHigh resolution crystal structures of the Escherichia coli lytic transglycosylase slt70 and its complex with a peptidoglycan fragment1
Introduction
Structural integrity of the bacterial cell wall is of vital importance for the prokaryotic cell. Without a rigid, intact cell wall, bacteria are not able to maintain their specific shapes. They may become spherical and burst as they are no longer able to resist the osmotic pressure inside their cells. The stress-resistant cell wall is, however, not a static structure: It has to increase in size to allow cell growth. Moreover, it is intimately involved in cell division, which requires precisely localised synthesis and cleavage of cell wall material Schwarz et al 1969, Romeis et al 1991. The simultaneous requirements for both rigidity and flexibility of the bacterial cell wall have been combined in a unique molecular meshwork known as murein or peptidoglycan. This is a biopolymer built up of linear glycan strands, that are cross-linked by short peptides, thus forming a network-like structure that surrounds the cytoplasmic membrane of the bacterial cell as one giant macromolecule (Weidel & Pelzer, 1964). Growth and division of the cell wall polymer are believed to result from a balanced action of murein-synthesising and murein-degrading enzymes (Höltje & Schwarz, 1985). In this finely tuned interplay of metabolic enzymes, the murein-degrading enzymes are considered to fulfil a number of fundamental tasks Shockman and Holtje 1994, Holtje 1998. Apart from taking care of specific cleavage of murein during cell division, they serve as “space makers” during cell growth, providing space and acceptor sites for incoming new murein material. In addition they form part of the peptidoglycan turnover and recycling system enabling the cell to make efficient use of old murein material for remodelling and enlargement of the cell wall. Unfortunately, knowledge of the precise biological functions of these enzymes, and of the mechanism by which the bacterium controls their lytic activity is still lacking.
Among the murein-degrading enzymes in the Gram-negative bacterium Escherichia coli, the lytic transglycosylases are of special interest. By cleaving the β-1,4-glycosidic bonds between the N-acetylmuramic acid (MurNAc) and N-acetyl-glucosamine (GlcNAc) residues of the glycan strands, these bacterial muramidases are able to totally degrade intact murein in vitro, as do various lysozymes. However, completely unlike lysozymes, they catalyse, concomitantly to cleavage, the synthesis of a new intramolecular glycosidic bond between carbons 1 and 6 of the MurNAc residue thereby forming a non-reducing 1,6-anhydro-N-acetylmuramic acid (anhMurNAc, Figure 1) (Höltje et al., 1975). Six different lytic transglycosylases have been isolated from E. coli. One is a soluble enzyme located in the periplasmic space with molecular mass of 70 kDa (Slt70) (Engel et al., 1991), the other five enzymes are membrane-bound lipo-proteins (MltA-D, EmtA) Engel et al 1992, Ursinus and Holtje 1994, Dijkstra et al 1995, Ehlert et al 1995, Dijkstra 1997, Lommatzsch et al 1997, Kraft et al 1998. They differ in their substrate preferences, in their enzymatic activity and in their susceptibilities towards certain glycopeptide inhibitors (Romeis et al., 1993). Slt70 has been characterised in most detail. The gene of Slt70 has been cloned and sequenced Betzner and Keck 1989, Engel et al 1991 and it encodes a monomeric enzyme of 618 amino acid residues. The enzyme acts as an exo-muramidase that removes disaccharide-peptides from one end of the glycan strands (Beachey et al., 1981) and it is inhibited by the antibiotic bulgecin A (Templin et al., 1992). The combined treatment of E. coli cells with this glycopeptide inhibitor and the β-lactam furazlocillin leads to the formation of bulges and enhanced bacteriolysis.
Considering their unique reaction and intriguing role in murein metabolism, lytic transglycosylases form highly interesting candidates for detailed structural studies. Moreover, there is a growing pharmaceutical interest in these enzymes. Since their activity is directed towards the glycosidic bonds in the glycan strands, they offer a target for structure-based design of novel antibiotics distinctly different from the penicillins and related β-lactam antibacterials, whose main target form the enzymes involved in the synthesis and cleavage of the peptide linkage between the glycan strands (Waxman & Strominger, 1983). A second interest is related to their 1,6-anhydromuropeptide products, which show a number of unusual biological activities in mammals, ranging from the ability to induce sleep Martin et al 1984, Johannsen 1993, to stimulate the immune response (Dokter et al., 1994) and to properties that are related to the pathogenesis of Gram-negative bacteria like Neisseria gonorrhoeae and Bordetella pertussis Melly et al 1984, Cookson et al 1989. The molecular basis for the activities shown by the 1,6-anhydromuropeptides is still not understood.
To increase our knowledge about the peptidoglycan-degrading enzymes, we study the three-dimensional structure of the periplasmic 70 kDa soluble lytic transglycosylase (Slt70) from E. coli with X-ray crystallography. The results have already provided information about the overall structure at 2.7 Å resolution and aspects of the catalytic function of this enzyme Thunnissen et al 1994, Thunnissen et al 1995a, Thunnissen et al 1995b. Here we report the refinement and a detailed analysis of its crystal structure at a nominal resolution 1.65 Å. Moreover, the crystal structure of Slt70 complexed with a 1,6-anhydro-muropeptide has been determined, revealing the presence of a peptide-binding site at the interface of the α-superhelix and the catalytic domain.
Section snippets
Refinement of models
The native Slt70 model has been refined at 1.65 Å resolution starting from the 2.7 Å resolution structure previously solved by Thunnissen et al. (1994). The high solvent content (about 63 %) and the large amount of electron-rich sulphate ions in the Slt70 crystal prompted the application of a bulk solvent correction to the lower resolution data during the refinement. Furthermore, the anisotropic scattering of the Slt70 crystal suggested the application of an overall anisotropic B-factor scaling
Origin and function of the α-superhelix
The α-superhelical U-domain forms the most remarkable feature of the Slt70 structure. The non-globular fold of this domain is not commonly observed for a single polypeptide chain. Stabilisation of the fold seems nevertheless to arise from the same principles that are believed to function in globular proteins. The main stabilisation comes from the burying of an extensive, though narrow core of mainly apolar residues. Alanine and leucine residues are found most often in the buried core of the
Crystallisation, data collection and processing
Purified Slt70 Betzner and Keck 1989, Engel et al 1991 was kindly provided by W. Keck and A. J. Dijkstra, Hoffmann-La Roche, Basel. Crystals of Slt70 were obtained by the hanging drop method using a macroscopic seeding technique (Rozeboom et al., 1990). Briefly, small, single crystals (average dimensions 0.1 mm × 0.05 mm × 0.05 mm), grown from previous crystallisation trials, were selected as seeds. The seeds were carefully washed in a 20 % (w/v) saturated ammonium sulphate solution in 0.1 M
Acknowledgements
We thank M. Szebenyi and D. Thiel for their help during data collection at the A1 station in CHESS, and A.J. Dijkstra and W. Keck (F. Hoffmann-La Roche Ltd., Basel) for the supply of protein and 1,6-anhydromuropeptide, and stimulating discussions. E.J.v.A. and A.M.W.H.T. contributed equally to the work reported in this paper. This work was supported by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for Scientific Research (NWO).
References (80)
- et al.
Crystal structure of glucoamylase from Aspergillus awamori var. X100 to 2.2-Å resolution
J. Bacteriol
(1992) - et al.
The structural basis of lipid interactions in lipovitellin, a soluble lipoprotein
Structure
(1998) - et al.
Conformational analysis and computer modelling of muramic acid δ-lactam structures
Carbohydr. Res
(1994) - et al.
Helix to helix packing in proteins
J. Mol. Biol
(1981) - et al.
Structures and mechanisms of glycosyl hydrolases
Structure
(1995) - et al.
Cloning and controlled overexpression of the gene encoding the 35 kDa soluble lytic transglycosylase from Escherichia Coli
FEBS Letters
(1995) - et al.
G(Anh)MTetra, a natural bacterial cell wall breakdown product, induces interleukin-1 beta and interleukin-6 expression in human monocytes. A study of the molecular mechanisms involved in inflammatory cytokine expression
J. Biol. Chem
(1994) - et al.
The structure of the protein phosphatase 2A PR65/A subunit reveals the conformation of its 15 tandemly repeated HEAT motifs
Cell
(1999) - et al.
Refined structure of cytochrome b562 from Escherichia Coli at 1.4 Å resolution
J. Mol. Biol
(1995) The role of the α-helix dipole in protein function and structure
Prog. Biophys. Mol. Biol
(1985)
Three-dimensional structure of the armadillo repeat region of β-catenin
Cell
Surface, subunit interface and interior of oligomeric proteins
J. Mol. Biol
Turn up the HEAT
Structure
Nucleotide sequence and X-ray structure of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 in a maltose-dependent crystal form
J. Mol. Biol
Peptidoglycan as promoters of slow-wave sleep1. Structure of the sleep-promoting factor isolated from human urine
J. Biol. Chem
Processing of X-ray diffraction data collection in oscillation mode
Methods Enzymol
Murein chemistry of cell division in Escherichia Coli
Res. Microbiol
Characterization of three different lytic transglycosylases in Escherichia Coli
FEMS Microbiol. Letters
Crystallization of the soluble lytic transglycosylase from Escherichia Coli K12
J. Mol. Biol
Autolytic enzymes and cell division of Escherichia Coli
J. Mol. Biol
SHELXLhigh resolution refinement
Methods Enzymol
A murein hydrolase is the specific target of bulgecin in Escherichia Coli
J. Biol. Chem
Refinement of the influenza virus hemagglutinin by simulated annealing
J. Mol. Biol
Mechanism of catalysis by β-glycosyl hydrolases
Curr. Opin. Struct. Biol
Unusual structural features in the parallel β-helix in pectate lyases
Structure
Gapped BLAST and PHI-BLASTa new generation of protein database search programs
Nucl. Acids Res
ALSCRIPTa tool to format multiple sequence alignments
Protein Eng
Three-dimensional structure of the alkaline protease of Pseudomonas aeruginosaa two domain protein with a calcium binding parallel β-roll motif
EMBO J
Exoenzymatic activity of transglycosylase isolated from Escherichia Coli
Eur. J. Biochem
Molecular cloning, overexpression and mapping of the slt gene encoding the soluble lytic transglycosylase of Escherichia Coli
Mol. Gen. Genet
The complete genome sequence of Escherichia Coli K-12
Science
Collaborative Computational Project Number 4 - The CCP4 suiteprograms for protein crystallography
Acta Crystallog. sect. D
Principles that determine the structure of proteins
Annu. Rev. Biochem
Structure of importin-β bound to the IBB domain of importin-α
Nature
Biological activities and chemical composition of purified tracheal cytotoxin of Bordetella pertussis
Infect. Immun
The soluble lytic transglycosylase family of Escherichia Coli. In vitro activity versus in vivo function
Peptidoglycan as a barrier to transenvelope transport
J. Bacteriol
Cloning and expression of a murein hydrolase lipoprotein from Escherichia Coli
Mol. Microbiol
Improved strategy in analytic surface calculation for molecular systemshandling of singularities and computational efficiency
J. Comp. Chem
Murein-metabolizing enzymes from Escherichia Colisequence analysis and controlled overexpression of the slt gene, which encodes the soluble lytic transglycosylase
J. Bacteriol
Cited by (0)
- 1
Edited by K. Nagai