Bacterial spores are the most resistant form of life known on Earth and represent a serious problem for (i) bioterrorism attack, (ii) horizontal transmission of microbial pathogens in the community, and (iii) persistence in patients and in a nosocomial environment. Stage II sporulation protein D (SpoIID) is a lytic transglycosylase (LT) essential for sporulation. The LT superfamily is a potential drug target because it is active in essential bacterial processes involving the peptidoglycan, which is unique to bacteria. However, the absence of structural information for the sporulation-specific LT enzymes has hindered mechanistic understanding of SpoIID. Here, we report the first crystal structures with and without ligands of the SpoIID family from two community relevant spore-forming pathogens, Bacillus anthracis and Clostridium difficile. The structures allow us to visualize the overall architecture, characterize the substrate recognition model, identify critical residues, and provide the structural basis for catalysis by this new family of enzymes.Sporulation is an evolutionarily conserved process that allows some Gram-positive bacteria to differentiate into a dormant cell type called a spore that allows them to resist chemical agents such as antibiotics and disinfectants and physical agents such as radiation, boiling and drying (1). Sporulation is characterized by an asymmetric cell division, followed by migration of the mother cells' membrane around the nascent spore in a phagocyte-like process (2, 3). Engulfment of what will become the spore requires PGN 2 degradation by the SpoIIM/SpoIIP/ SpoIID molecular machine (4, 5). In this complex, SpoIIM serves as a scaffold upon which the SpoIID and SpoIIP enzymes assemble (1, 4, 6). SpoIIP is single-pass transmembrane protein with a large extracellular portion that has both amidase and endopeptidase activities. This enzyme cleaves the cross-links connecting the stem peptides of PGN and removes the stem peptides from the glycan chains (Fig. 1a). SpoIID is a lytic transglycosylase (LT), an enzyme that cleaves the glycan strands of PGN (Fig. 1b). It also has a single-pass transmembrane domain and a large extracellular portion that can bind PGN but can only cleave glycan strands after the stem peptides of PGN have been removed by SpoIIP. The requirement for a peptide-free glycan strand defines an ordered and sequential set of events during the engulfment process. SpoIID also functions to stimulate SpoIIP activity (4).LTs represent a class of autolysin that cleaves the glycan chain of the PGN to facilitate biosynthesis, remodeling, degradation, and turnover of the bacterial cell wall (7). LTs differ from the other two classes of glycan strand-cleaving enzymes. Although muramidases hydrolyze the -1,4 N-acetylmuramic acid (NAM)-N-acetylglucosamine (NAG) glycosidic bond and glucosaminidases hydrolyze the -1,4 NAG-NAM glycosidic bond, LTs use the C-6 hydroxyl of NAM as a nucleophile in the cleavage reaction of the -1,4 NAM-NAG glycosidic bond. Consequently, the product o...
