Estrogens are known modulators of monocyte/macrophage functions; however, the underlying mechanism has not been clearly defined. Recently, a number of estrogen receptor molecules and splice variants were identified that exert different and sometimes opposing actions. We assessed the expression of estrogen receptors and explored their role in mediating estrogenic anti-inflammatory effects on human primary monocytes. We report that the only estrogen receptors expressed are estrogen receptor-α 36-kDa splice variant and G-protein coupled receptor 30/G-protein estrogen receptor 1, in a sex-independent manner. 17-β-Estradiol inhibits the LPS-induced IL-6 inflammatory response, resulting in inhibition of NF-κB transcriptional activity. This is achieved via a direct physical interaction of ligand-activated estrogen receptor-α 36-kDa splice variant with the p65 component of NF-κB in the nucleus. G-protein coupled receptor 30/G-protein estrogen receptor 1, which also physically interacts with estrogen receptor-α 36-kDa splice variant, acts a coregulator in this process, because its inhibition blocks the effect of estrogens on IL-6 expression. However, its activation does not mimic the effect of estrogens, on neither IL-6 nor NF-κB activity. Finally, we show that the estrogen receptor profile observed in monocytes is not modified during their differentiation to macrophages or dendritic cells in vitro and is shared in vivo by macrophages present in atherosclerotic plaques. These results position estrogen receptor-α 36-kDa splice variant and G-protein coupled receptor 30 as important players and potential therapeutic targets in monocyte/macrophage-dependent inflammatory processes.

The genomes of Bacillus cereus and its closest relative Bacillus anthracis contain 10 polysaccharide deacetylase homologues. Six of these homologues have been proposed to be peptidoglycan N-acetylglucosamine deacetylases. Two of these genes, namely bc1960 and bc3618, have been cloned and expressed in Escherichia coli, and the recombinant enzymes have been purified to homogeneity and further characterized. Both enzymes were effective in deacetylating cell wall peptidoglycan from the Gram(؉) Bacillus cereus and Bacillus subtilis and the Gram(؊) Helicobacter pylori as well as soluble chitin substrates and N-acetylchitooligomers. However, the enzymes were not active on acetylated xylan. These results provide insight into the substrate specificity of carbohydrate esterase family 4 enzymes. It was revealed that both enzymes deacetylated only the GlcNAc residue of the synthetic muropeptide N-acetyl-D-glucosamine-(␤-1,4)-N-acetylmuramyl-L-alanine-D-isoglutamine. Analysis of the constituent muropeptides of peptidoglycan from B. subtilis and H. pylori resulting from incubation of the enzymes BC1960 and BC3618 with these polymers and subsequent hydrolysis by Cellosyl and mutanolysin, respectively, similarly revealed that both enzymes deacetylate GlcNAc residues of peptidoglycan. Kinetic analysis toward GlcNAc 2-6 revealed that GlcNAc 4 was the favorable substrate for both enzymes. Identification of the sequence of N-acetychitooligosaccharides (GlcNAc 2-4 ) following enzymatic deacetylation by using 1 H NMR revealed that both enzymes deacetylate all GlcNAc residues of the oligomers except the reducing end ones. Enzymatic deacetylation of chemically acetylated vegetative peptidoglycan from B. cereus by BC1960 and BC3618 resulted in increased resistance to lysozyme digestion. This is the first biochemical study of bacterial peptidoglycan N-acetylglucosamine deacetylases.

Bacillus cereus, an opportunistic pathogen that causes food poisoning and Bacillus antracis, the endosporeforming bacterium that causes inhalational anthrax, share a large number of homologous genes, as demonstrated by the recent genome sequencing and comparative analysis [1,2]. Given the laboratory safety precautions necessary for working with highly infectious agents and the recent concerns related to B. anthracis as a potential bioweapon, B. cereus offers an attractive alternative for studying the corresponding proteins of B. anthracis because it lacks infectiousness of the latter. The objective of the present study is to shed light on the structure, function and the structurefunction relationships of one B. cereus protein, a product of the bc1534 gene, which is highly conserved among the two pathogens and which has, as we show, acetylchitooligosaccharide deacetylase activity. Thus, our work contributes to the understanding of the role Bacillus cereus is an opportunistic pathogenic bacterium closely related to Bacillus anthracis, the causative agent of anthrax in mammals. A significant portion of the B. cereus chromosomal genes are common to B. anthracis, including genes which in B. anthracis code for putative virulence and surface proteins. B. cereus thus provides a convenient model organism for studying proteins potentially associated with the pathogenicity of the highly infectious B. anthracis. The zinc-binding protein of B. cereus, BcZBP, is encoded from the bc1534 gene which has three homologues to B. anthracis. The protein exhibits deacetylase activity with the N-acetyl moiety of the N-acetylglucosamine and the diacetylchitobiose and triacetylchitotriose. However, neither the specific substrate of the BcZBP nor the biochemical pathway have been conclusively identified. Here, we present the crystal structure of BcZBP at 1.8 Å resolution. The N-terminal part of the 234 amino acid protein adopts a Rossmann fold whereas the C-terminal part consists of two b-strands and two a-helices. In the crystal, the protein forms a compact hexamer, in agreement with solution data. A zinc binding site and a potential active site have been identified in each monomer. These sites have extensive similarities to those found in two known zinc-dependent hydrolases with deacetylase activity, MshB and LpxC, despite a low degree of amino acid sequence identity. The functional implications and a possible catalytic mechanism are discussed.

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.

Psychrophilic alkaline phosphatase (AP) from the Antarctic strain TAB5 was subjected to directed evolution in order to identify the key residues steering the enzyme's cold-adapted activity and stability. A round of random mutagenesis and further recombination yielded three thermostable and six thermolabile variants of the TAB5 AP. All of the isolated variants were characterised by their residual activity after heat treatment, Michaelis-Menten kinetics, activation energy and microcalorimetric parameters of unfolding. In addition, they were modelled into the structure of the TAB5 AP. Mutations which affected the cold-adapted properties of the enzyme were all located close to the active site. The destabilised variants H135E and H135E/G149D had 2- and 3-fold higher kcat, respectively, than the wild-type enzyme. Wild-type AP has a complex heat-induced unfolding pattern while the mutated enzymes loose local unfolding transitions and have large shifts of the Tm values. Comparison of the wild-type and mutated TAB5 APs demonstrates that there is a delicate balance between the enzyme activity and stability and that it is possible to improve the activity and thermostability simultaneously as demonstrated in the case of the H135E/G149D variant compared to H135E.