Streptococcus pneumoniae peptidoglycan GlcNAc deacetylase (SpPgdA) protects the Gram-positive bacterial cell wall from host lysozymes by deacetylating peptidoglycan GlcNAc residues. Deletion of the pgda gene has been shown to result in hypersensitivity to lysozyme and reduction of infectivity in a mouse model. SpPgdA is a member of the family 4 carbohydrate esterases, for which little structural information exists, and no catalytic mechanism has yet been defined. Here we describe the native crystal structure and product complexes of SpPgdA biochemical characterization and mutagenesis. The structural data show that SpPgdA is an elongated three-domain protein in the crystal. The structure, in combination with mutagenesis, shows that SpPgdA is a metalloenzyme using a His-His-Asp zinc-binding triad with a nearby aspartic acid and histidine acting as the catalytic base and acid, respectively, somewhat similar to other zinc deacetylases such as LpxC. The enzyme is able to accept GlcNAc 3 as a substrate (Km ‫؍‬ 3.8 mM, kcat ‫؍‬ 0.55 s ؊1 ), with the N-acetyl of the middle sugar being removed by the enzyme. The data described here show that SpPgdA and the other family 4 carbohydrate esterases are metalloenzymes and present a step toward identification of mechanismbased inhibitors for this important class of enzymes.crystal structure ͉ metalloenzyme

The fungal pathogen Colletotrichum lindemuthianum secretes an endo-chitin de-N-acetylase (ClCDA) to modify exposed hyphal chitin during penetration and infection of plants. Although a significant amount of biochemical data is available on fungal chitin de-N-acetylases, no structural data exist. Here we describe the 1.8 Å crystal structure of a ClCDA product complex and the analysis of the reaction mechanism using Hammett linear free energy relationships, subsite probing, and atomic absorption spectroscopy studies. The structural data in combination with biochemical data reveal that ClCDA consists of a single domain encompassing a mononuclear metalloenzyme which employs a conserved His-HisAsp zinc-binding triad closely associated with the conserved catalytic base (aspartic acid) and acid (histidine) to carry out acid/base catalysis. The data presented here indicate that ClCDA possesses a highly conserved substrate-binding groove, with subtle alterations that influence substrate specificity and subsite affinity. Strikingly, the structure also shows that the hexahistidine purification tag appears to form a tight interaction with the active site groove. The enzyme requires occupancy of at least the 0 and +1 subsites by (GlcNAc) 2 for activity and proceeds through a tetrahedral oxyanion intermediate.Colletotrichum lindemuthianum is a plant fungal pathogen found extensively in tropical and subtropical regions. Colletotrichum species are the causative agent of anthracnose that affects economically important crop species (1). Furthermore, Colletotrichum sp. have recently been reported to cause subcutaneous and systemic infections among immunosuppressed patients (2). Colletotrichum sp. are facultative biotrophs. Before the fungal hyphae can successfully penetrate and gain access to host tissue, the fungus has to first evade plant antimicrobial hydrolases such as chitinases and -(1,3)glucanases (3, 4). The chitinases degrade fungal chitin, an insoluble linear polymer of -(1-4)-linked N-acetylglucosamine (GlcNAc). 1 The breakdown products may act as elicitors of active defense responses within the plant (5-7). Studies of cell wall composition of invasive fungal hyphae suggest that exposed fungal chitin polymers are partially de-N-acetylated during infection and initial growth within the host (8). Chitosan, the de-N-acetylated product, is a poor substrate for chitinases, which require the presence of N-acetyl moieties for recognition and catalysis (9). Thus, conversion of chitin to chitosan during plant extracellular colonization may protect pathogenic fungal hyphae from being lysed by secreted plant chitinases. The enzyme responsible for chitin modification is a developmentally regulated, secreted, chitin deacetylase (CDA) (10). C. lindemuthianum chitin deacetylase (ClCDA) is a member of the family 4 carbohydrate esterases (CE-4s) as defined by the CAZY database [http://afmb.cnrs-mrs.fr/∼cazy/CAZY (11)], which include several members that share the primary structure assigned as the "NodB homology domain" (12). Rh...

Protein O-GlcNAcylation is an essential post-translational modification on hundreds of intracellular proteins in metazoa, catalyzed by O-GlcNAc transferase using unknown mechanisms of transfer and substrate recognition. Through crystallographic snapshots and mechanism-inspired chemical probes, we define how human O-GlcNAc transferase recognizes the sugar donor and acceptor peptide and employs a novel catalytic mechanism of glycosyl transfer, involving the sugar donor α-phosphate as the catalytic base, as well as an essential lysine. This mechanism appears to be a unique evolutionary solution to the spatial constraints imposed by a bulky protein acceptor substrate, and explains the unexpected specificity of a recently reported metabolic O-GlcNAc transferase inhibitor.