DNA methyltransferase M. Bse CI from Geobacillus stearothermophilus (EC 2.1.1.72), an enzyme with 579 residues, methylates the N6 atom of the 3'-adenine in the sequence 5'-ATCGAT-3'. Here we report the overexpression, purification and cocrystallization of M. Bse CI with the cofactor S -adenosyl-L-homocysteine and its cognate DNA sequence in two different methylation states, the hemi- and fully methylated states, corresponding to the natural substrate and the final product of the methylation reaction. For a thermophilic enzyme, effective complex formation with DNA was achieved only at elevated temperatures. Combined with our previous studies, this provides an opportunity to clarify the structural basis of methylation in adenine-specific methyltransferases. Crystals were found to belong to the hexagonal space group P 6 1 with the unit–cell parameters a = b = 87.7 Å, c = 156.1 Å, γ = 120° for the complex with the hemimethylated DNA and a = b = 88.0 Å, c = 155.5 Å, γ = 120° for the complex with the fully methylated DNA. For both crystals, complete initial data sets were collected, extending to 2.4 Å for the hemimethylated and to 2.6 Å for the fully methylated complex.

The structure of a recombinant (His-tagged at C-terminus) alcohol dehydrogenase (MoADH) from the cold-adapted bacterium Moraxella sp. TAE123 has been refined with X-ray diffraction data extending to 1.9 Å resolution. The enzyme assumes a homo-tetrameric structure. Each subunit comprises two distinct structural domains: the catalytic domain (residues 1-150 and 288-340/345) and the nucleotide-binding domain (residues 151-287). There are two Zn2+ ions in each protein subunit. Two additional zinc ions have been found in the crystal structure between symmetry-related subunits. The structure has been compared with those of homologous enzymes from Geobacillus stearothermophilus (GsADH), Escherichia coli (EcADH), and Thermus sp. ATN1 (ThADH) that thrive in environments of diverse temperatures. Unexpectedly, MoADH has been found active from 10 to at least 53 °C and unfolds at 89 °C according to circular dichroism spectropolarimetry data. MoADH with substrate ethanol exhibits a small value of activation enthalpy ΔH ‡ of 30 kJ mol-1. Molecular dynamics simulations for single subunits of the closely homologous enzymes MoADH and GsADH performed at 280, 310, and 340 K showed enhanced wide-ranging mobility of MoADH at high temperatures and generally lower but more distinct and localized mobility for GsADH. Principal component analysis of the fluctuations of both ADHs resulted in a prominent open-close transition of the structural domains mainly at 280 K for MoADH and 340 K for GsADH. In conclusion, MoADH is a very thermostable, cold-adapted enzyme and the small value of activation enthalpy allows the enzyme to function adequately at low temperatures.

Putative and known polysaccharide deacetylases (PDAs) from B. anthracis have key roles in resistance to host lysozyme, stabilization of the cell wall, biogenesis of peptidoglycan (PG) and for neutral polysaccharide modification and attachment to PG. Here we elucidated the physiological role of the putative PDA BA1836 from B. anthracis. The ba1836 gene was expressed upon entrance into the stationary phase of growth and enhanced during the early stages of sporulation. The Δba1836 knockout strain had normal growth rate, did not exhibit any significant alterations in PG pattern of stationary phase cells and was not sensitive to lysozyme, but showed a defect in cell separation. Strikingly, the Δba1836 mutant strain exhibited a severe delay in spore development although mature spores were ultimately developed and had normal morphology. Additionally, digestion of Δba1836 mutant spore PG with mutanolysin produced an almost identical muropeptide pattern compared to peptidoglycan from wild type spores, although the amount of all muropeptides was significantly reduced. Finally, knockout spores exhibited a lower germination rate. To our knowledge, BA1836 has a unique role, among the presently characterized PDAs from B. anthracis, in spore development and germination.

Ba0331 is a putative polysaccharide deacetylase from Bacillus anthracis, the etiological agent of the disease anthrax, that contributes to adaptation of the bacterium under extreme conditions and to maintenance of the cell shape. In the present study, the crystal structure of Ba0331 was determined at 2.6 Å resolution. The structure consists of two domains: a fibronectin type 3-like (Fn3-like) domain and a NodB catalytic domain. The latter is present in all carbohydrate esterase family 4 enzymes, while a comparative analysis of the Fn3-like domain revealed structural plasticity despite the retention of the conserved Fn3-like domain characteristics.

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.

Peptidoglycan N-acetylglucosamine (GlcNAc) deacetylases (PGNGdacs) from bacterial pathogens are validated targets for the development of novel antimicrobial agents. In this study we examined the in vitro inhibition of hydroxamate ligand N-hydroxy-4-(naphthalene-1-yl)benzamide (NHNB), a selective inhibitor of histone deacetylases-8 (HDAC8), against two PGNGdacs namely BC1974 and BC1960 from B. cereus, highly homologous to BA1977 and BA1961 of B. anthracis, respectively. Kinetic analysis showed that this compound functions as a competitive inhibitor of both enzymes with apparent Ki's of 8.7 μM (for BC1974) and 66 μM (for BC1960), providing thus the most potent CE4 inhibitor reported to date. NHNB was tested in antibacterial assays and showed bactericidal activity against both examined pathogens acting as a multi-target drug. This compound can serve as lead for the development of inhibitors targeting the conserved active sites of the multiple polysaccharide deacetylases (PDAs) of both pathogens.

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 Cα 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.

There is an urgent need to explore new targets and develop novel therapeutics due to the emergence of multi-drug resistant bacteria. Recently, selected members of Carbohydrate Esterase family 4 namely peptidoglycan and exopolysaccharide de-N-acetylases from bacterial pathogens have been identified as promising antibacterial drug targets. This chapter highlights the state of the art in their physiological roles, biochemical characterization, regulation, localization, structure determination and mechanism of action. Initial efforts and strategies that will guide the development of novel inhibitors against bacterial pathogens, including biofilm forming and spore forming ones, are also reviewed.

Background: BA0330 and BA0331 are the only two lipoproteins among 11 known or putative polysaccharide deacetylases from Bacillus anthracis. Results: Both proteins lack deacetylase activity and are important for cell shape maintenance (BA0331) or high salt stress adaptation of the bacterium (BA0330). Conclusion: BA0330 and BA0331 stabilize the cell wall of B. anthracis. Significance: Understanding the mechanisms by which lipoproteins maintain cell wall integrity. Membrane-anchored lipoproteins have a broad range of functions and play key roles in several cellular processes in Gram-positive bacteria. BA0330 and BA0331 are the only lipoproteins among the 11 known or putative polysaccharide deacetylases of Bacillus anthracis. We found that both lipoproteins exhibit unique characteristics. BA0330 and BA0331 interact with peptidoglycan, and BA0330 is important for the adaptation of the bacterium to grow in the presence of a high concentration of salt, whereas BA0331 contributes to the maintenance of a uniform cell shape. They appear not to alter the peptidoglycan structure and do not contribute to lysozyme resistance. The high resolution x-ray structure of BA0330 revealed a C-terminal domain with the typical fold of a carbohydrate esterase 4 and an N-terminal domain unique for this family, composed of a two-layered (4 + 3) β-sandwich with structural similarity to fibronectin type 3 domains. Our data suggest that BA0330 and BA0331 have a structural role in stabilizing the cell wall of B. anthracis.

Bacillus anthracis has a complex cell wall structure composed of a peptidoglycan (PG) layer to which major structures are anchored such as a neutral polysaccharide, an S-layer, and a poly-γ-D-glutamate (PDGA) capsule. Many of these structures have central roles in the biology of B. anthracis, particularly, in virulence. However, little attention has been devoted to structurally study the PG and how it is modified in the presence of these secondary cell wall components. We present here the fine structure of the PG of the encapsulated RPG1 strain harboring both pXO1 and pXO2 virulence plasmids. We show that B. anthracis has a high degree of cross-linking and its GlcNAc residues are highly modified by N-deacetylation. The PG composition is not dependent on the presence of either LPXTG proteins or the capsule. Using NMR analysis of the PG-PDGA complex, we provide evidence for the anchoring of the PDGA to the glucosamine residues. We show that anchoring of the PDGA capsule is impaired in two PG N-deacetylase mutants, Ba1961 and Ba3679. Thus, these multiple N-deactylase activities would constitute excellent drug targets in B. anthracis by simultaneously affecting its resistance to lysozyme and to phagocytosis impairing B. anthracis survival in the host.

Polysaccharide deacetylases are bacterial enzymes that catalyze the deacetylation of acetylated sugars on the membranes of Gram-positive bacteria, allowing them to be unrecognized by host immune systems. Inhibition of these enzymes would disrupt such pathogenic defensive mechanisms and therefore offers a promising route for the development of novel antibiotic therapeutics. Here, the first X-ray crystal structure of BA0150, a putative polysaccharide deacetylase from Bacillus anthracis, is reported to 2.0 Å resolution. The overall structure maintains the conserved (α/β)8 fold that is characteristic of this family of enzymes. The lack of a catalytic metal ion and a distinctive metal-binding site, however, suggest that this enzyme is not a functional polysaccharide deacetylase.

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 structure of BC0361, a polysaccharide deacetylase from Bacillus cereus, has been determined using an unconventional molecular-replacement procedure. Tens of putative models of the C-terminal domain of the protein were constructed using a multitude of homology-modelling algorithms, and these were tested for the presence of signal in molecular-replacement calculations. Of these, only the model calculated by the SAM-T08 server gave a consistent and convincing solution, but the resulting model was too inaccurate to allow phase determination to proceed to completion. The application of slow-cooling torsion-angle simulated annealing (started from a very high temperature) drastically improved this initial model to the point of allowing phasing through cycles of model building and refinement to be initiated. The structure of the protein is presented with emphasis on the presence of a C(α)-modified proline at its active site, which was modelled as an α-hydroxy-L-proline.

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-alpha-d-glucopyranoside (GlcNAc-Ins), N,N'-diacetylchitobiose (GlcNAc(2)) 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 (GlcNAc(2), GlcNAc(3) and GlcNAc(4)), 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-(beta-1,4)-N-acetylmuramyl-l-alanyl-d-isoglutamine (GMDP) or N-acetyl-d-GlcN-N alpha 1-6-d-myo-inositol-1-HPO4-octadecyl (GlcNAc-I-P-C-18). Kinetic analysis of the activity of BC1534 and BC3461 on GlcNAc and GlcNAc(2) revealed that GlcNAc(2) 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.