Abstract
The transpeptidase LtdMt2 catalyzes the formation of the (3-3) cross-links characteristic of the peptidoglycan layer in the Mycobacterium tuberculosis cell wall. Bioinformatics analysis suggests that the extramembrane part of the enzyme consists of three domains: two smaller domains (denoted as A and B domains) and a transpeptidase domain (the C domain) at the C-terminus. The crystal structures of two fragments comprising the AB domains and the BC domains have been determined. The structure of the BC module, which was determined to 1.86 Å resolution using Se-SAD phasing, consists of the B domain with an immunoglobulin-related fold and the catalytic domain belonging to the ErfK/YbiS/YbnG fold family. The structure of the AB-domain fragment, which was solved by molecular replacement to 1.45 Å resolution, reveals that despite a lack of overall sequence identity the A domain is structurally very similar to the B domain. Combining the structures of the two fragments provides a view of the complete three-domain extramembrane part of LdtMt2 and shows that the protein extends at least 80-100 Å from the plasma membrane into the peptidoglycan layer and thus defines the maximal distance at which cross-links are formed by this enzyme. The LdtMt-related transpeptidases contain one or two immunoglobulin domains, which suggests that these might serve as extender units to position the catalytic domain at an appropriate distance from the membrane in the peptidoglycan layer.
Highlights
The cell wall of Mycobacterium tuberculosis is a complex multilayered structure with a unique architecture that protects the organism against both physical and chemical stress (Hett & Rubin, 2008)
We report the three-dimensional structure of LdtMt2 (l,d-transpeptidase 2 from M. tuberculosis) based on the X-ray crystallographic structures of two fragments of LdtMt2 representing the extramembrane part of the protein
X-ray data sets were collected to 1.45 Aresolution for the AB module and to 1.86 Aresolution for the BC module on beamline ID14-1 of the European Synchrotron Radiation Facility (ESRF), Grenoble, France
Summary
The cell wall of Mycobacterium tuberculosis is a complex multilayered structure with a unique architecture that protects the organism against both physical and chemical stress (Hett & Rubin, 2008). M. tuberculosis displays a common peptide-stem sequence (l-Ala-d- Glu-Dap-d-Ala) which is found in Escherichia coli and Bacillus subtilis, but with characteristic modifications These are the high level of amidation on the d-glutamate (d-Glu) and diaminopimelate (Dap) moieties and a high proportion (80%) of Dap–Dap (3–3) cross-links (Kumar et al, 2012; Lavollay et al, 2008). The crystal structures of the LdtMt2 constructs allow modelling of the full-length extramembrane part of the enzyme (residues 55–408), providing an estimate of the maximal distance of the catalytic site from the membrane and thereby the approximate distance at which 3–3 cross-links are formed in the PG layer. Mass-spectrometric analysis provides further evidence that LdtMt2 forms covalent adducts with the -lactam antibiotics imipenem and ampicillin
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