Purification, crystallization and preliminary characterization of a putative LmbE-like deacetylase from<i>Bacillus cereus</i>

Fadouloglou, Vasiliki E.; Kotsifaki, Dina; Gazi, Anastasia D.; Fellas, Georgios; Meramveliotaki, C.; Deli, Alexandra; Psylinakis, Emmanuel; Bouriotis, Vassilis; Kokkinidis, Michael

doi:10.1107/s1744309106004660

Cited by 4 publications

(5 citation statements)

References 11 publications

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“…5A). The positions of these bands are in agreement with the molecular mass of purified BC1534 estimated by size‐exclusion chromatography [13] and glutaraldehyde cross‐linking [18] (approximately 160 kDa), indicating that the protein exists as a hexamer in solution.…”

Section: Resultssupporting

confidence: 77%

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LmbE proteins from Bacillus cereus are de‐N‐acetylases with broad substrate specificity and are highly similar to proteins in Bacillus anthracis

et al. 2010

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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.

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Section: Resultssupporting

confidence: 77%

“…In the crystal, the protein forms a compact hexamer (Fig. 3A), in agreement with solution data [13]. A zinc binding site and a potential active site have been identified in each monomer.…”

Section: Introductionsupporting

confidence: 82%

LmbE proteins from Bacillus cereus are de‐N‐acetylases with broad substrate specificity and are highly similar to proteins in Bacillus anthracis

et al. 2010

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“…These values indicate a significant stabilization upon hexamerization. In agreement with the crystal structure, gel filtration experiments have shown that the enzyme elutes as a single peak to a volume that is consistent with a spherical hexamer [17]. These results for Bc ZBP are consistent with an ultracentrifugation analysis of TT1542 [7], which also indicate a hexameric assembly.…”

Section: Resultssupporting

confidence: 75%

“…The expression, purification and crystallization of Bc ZBP have been reported previously [17]. High resolution diffraction data were collected from a single frozen crystal (100 K) using beamline X12 at the European Molecular Biology Laboratory/Deutsches Elektronen‐Synchrotron (Hamburg, Germany).…”

Section: Methodsmentioning

confidence: 99%

Crystal structure of the BcZBP, a zinc‐binding protein from Bacillus cereus

et al. 2007

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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.

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“…Part of this interest was subsequently transferred to more benign, but still closely related to B. anthracis , species like B. cereus , an opportunistic bacterium causing food poisoning . In a drive to characterize, both functionally and structurally, deacetylases that are shared between these two organisms (and which may be implicated in metabolic pathways of biotechnological and pharmaceutical interest), we have recently reported the characterization, purification, crystallization and crystal structure determination of BcZBP , a homohexameric, LmbE-like, zinc-dependent deacetylase from B. cereus . − BcZBP is the product of the bc1534 gene, with three, thus far uncharacterized, homologues in B. anthracis sharing sequence identities (at the protein level) of 96%, 28%, and 24%, respectively. Functional studies showed that BcZBP preferentially deacetylates N -acetylglucosamine and diacetylchitobiose, although its specific substrate remains unknown.…”

Section: Introductionmentioning

confidence: 99%

Molecular Dynamics Simulations of BcZBP, A Deacetylase from Bacillus cereus: Active Site Loops Determine Substrate Accessibility and Specificity

Fadouloglou

Stavrakoudis

Bouriotis

et al. 2009

J. Chem. Theory Comput.

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BcZBP is an LmbE-like, homohexameric, zinc-dependent deacetylase from the opportunistic pathogen Bacillus cereus with three, thus far uncharacterized, homologues in B. anthracis. Although its specific substrate is still unknown, the enzyme has been shown to preferentially deacetylate N-acetylglucosamine and diacetylchitobiose via an active site based on a zinc-binding motif of the type HXDDXnH. In the crystal structure, the active site is located at a deep and partially blocked cleft formed at the interface between monomers related by the molecular 3-fold axis, although the major, in structural terms, building block of the enzyme is not the trimer, but the intertwined dimer. Here, we report results from a 50 ns molecular dynamics simulation of BcZBP in explicit solvent with full electrostatics and show that (i) the view of the intertwined dimer as the major structural and functional building block of this class of hexameric enzymes is possibly an oversimplification of the rather complex dynamics observed in the simulation, (ii) the most mobile (with respect to their atomic fluctuations) parts of the structure coincide with three surface loops surrounding the active site, and (iii) these mobile loops define the active site's accessibility, and may be implicated in the determination of the enzyme's specificity.

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Purification, crystallization and preliminary characterization of a putative LmbE-like deacetylase fromBacillus cereus

Cited by 4 publications

References 11 publications

LmbE proteins from Bacillus cereus are de‐N‐acetylases with broad substrate specificity and are highly similar to proteins in Bacillus anthracis

LmbE proteins from Bacillus cereus are de‐N‐acetylases with broad substrate specificity and are highly similar to proteins in Bacillus anthracis

Crystal structure of the BcZBP, a zinc‐binding protein from Bacillus cereus

Molecular Dynamics Simulations of BcZBP, A Deacetylase from Bacillus cereus: Active Site Loops Determine Substrate Accessibility and Specificity

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