SummaryPeptidoglycan deacetylases (PGNG-dacs) belong to the Carbohydrate Esterase Family 4 (CE4) and have been described as required for bacterial evasion to lysozyme and innate immune responses. Interestingly, there is an unusual occurrence of 10 putative polysaccharide deacetylases, including five PGNG-dacs, in the Bacillus sp. genomes, especially B. cereus and B. anthracis. To elucidate the physiological role of these multiple deacetylases, we employed genetic analysis and protein localization studies of five putative PGNG-dacs from B. anthracis as well as biochemical analysis of their corresponding homologues from B. cereus. Our data confirm that three enzymes are PGNG-dacs. While BA1977, associated with lateral peptidoglycan synthesis, is a bona fide peptidoglycan deacetylase involved in resistance to host lysozyme and required for full virulence, BA1961 and BA3679 participate in the biogenesis of the peptidoglycan during both elongation and cell division. Furthermore, two enzymes are important for neutral polysaccharide attachment to PG and consequently anchoring of S-layer proteins (BA5436) and for polysaccharide modification (BA2944). Our results provide novel and fundamental insights into the function of polysaccharide deacetylases in a major bioterrorism agent.

The genomes of Bacillus cereus and its closest relative Bacillus anthracis each contain two LmbE protein family homologs: BC1534 (BA1557) and BC3461 (BA3524). Only a few members of this family have been biochemically characterized including N‐acetylglucosaminylphosphatidyl inositol (GlcNAc‐PI), 1‐d‐myo‐inosityl‐2‐acetamido‐2‐deoxy‐α‐d‐glucopyranoside (GlcNAc‐Ins), N,N′‐diacetylchitobiose (GlcNAc2) and lipoglycopeptide antibiotic de‐N‐acetylases. All these enzymes share a common feature in that they de‐N‐acetylate the N‐acetyl‐d‐glucosamine (GlcNAc) moiety of their substrates. The bc1534 gene has previously been cloned and expressed in Escherichia coli. The recombinant enzyme was purified and its 3D structure determined. In this study, the bc3461 gene from B. cereus ATCC14579 was cloned and expressed in E. coli. The recombinant enzymes BC1534 (EC 3.5.1.‐) and BC3461 were biochemically characterized. The enzymes have different molecular masses, pH and temperature optima and broad substrate specificity, de‐N‐acetylating GlcNAc and N‐acetylchito‐oligomers (GlcNAc2, GlcNAc3 and GlcNAc4), as well as GlcNAc‐1P, N‐acetyl‐d‐glucosamine‐1 phosphate; GlcNAc‐6P, N‐acetyl‐d‐glucosamine‐6 phosphate; GalNAc, N‐acetyl‐d‐galactosamine; ManNAc, N‐acetyl‐d‐mannosamine; UDP‐GlcNAc, uridine 5′‐diphosphate N‐acetyl‐d‐glucosamine. However, the enzymes were not active on radiolabeled glycol chitin, peptidoglycan from B. cereus, N‐acetyl‐d‐glucosaminyl‐(β‐1,4)‐N‐acetylmuramyl‐l‐alanyl‐d‐isoglutamine (GMDP) or N‐acetyl‐d‐GlcN‐Nα1‐6‐d‐myo‐inositol‐1‐HPO4‐octadecyl (GlcNAc‐I‐P‐C18). Kinetic analysis of the activity of BC1534 and BC3461 on GlcNAc and GlcNAc2 revealed that GlcNAc2 is the favored substrate for both native enzymes. Based on the recently determined crystal structure of BC1534, a mutational analysis identified functional key residues, highlighting their importance for the catalytic mechanism and the substrate specificity of the enzyme. The catalytic efficiencies of BC1534 variants were significantly decreased compared to the native enzyme. An alignment‐based tree places both de‐N‐acetylases in functional categories that are different from those of other LmbE proteins.

The cell wall peptidoglycan is recognized as a primary target of the innate immune system, and usually its disintegration results in bacterial lysis. Bacillus cereus, a close relative of the highly virulent Bacillus anthracis, contains 10 polysaccharide deacetylases. Among these, the peptidoglycan N-acetylglucosamine deacetylase Bc1974 is the highest homologue to the Bacillus anthracis Ba1977 that is required for full virulence and is involved in resistance to the host's lysozyme. These metalloenzymes belong to the carbohydrate esterase family 4 (CE4) and are attractive targets for the development of new anti-infective agents. Herein we report the first X-ray crystal structures of the NodB domain of Bc1974, the conserved catalytic core of CE4s, in the unliganded form and in complex with four known metalloenzyme inhibitors and two amino acid hydroxamates that target the active site metal. These structures revealed the presence of two conformational states of a catalytic loop known as motif-4 (MT4), which were not observed previously for peptidoglycan deacetylases, but were recently shown in the structure of a Vibrio clolerae chitin deacetylase. By employing molecular docking of a substrate model, we describe a catalytic mechanism that probably involves initial binding of the substrate in a receptive, more open state of MT4 and optimal catalytic activity in the closed state of MT4, consistent with the previous observations. The ligand-bound structures presented here, in addition to the five Bc1974 inhibitors identified, provide a valuable basis for the design of antibacterial agents that target the peptidoglycan deacetylase Ba1977.

The full extent of proline (Pro) hydroxylation has yet to be established, as it is largely unexplored in bacteria. We describe here a so far unknown Pro hydroxylation activity which occurs in active sites of polysaccharide deacetylases (PDAs) from bacterial pathogens, modifying the protein backbone at the C atom of a Pro residue to produce 2-hydroxyproline (2-Hyp). This process modifies with high specificity a conserved Pro, shares with the deacetylation reaction the same active site and one catalytic residue, and utilizes molecular oxygen as source for the hydroxyl group oxygen of 2-Hyp. By providing additional hydrogen-bonding capacity, the Pro→2-Hyp conversion alters the active site and enhances significantly deacetylase activity, probably by creating a more favorable environment for transition-state stabilization. Our results classify this process as an active-site "maturation", which is highly atypical in being a protein backbone-modifying activity, rather than a side-chain-modifying one.