[Show abstract][Hide abstract] ABSTRACT: In the environment, vanadium-dependent haloperoxidases (VHPO) are likely to play a key role in the production of biogenic organo-halogens. These enzymes contain vanadate as a prosthetic group, and catalyze, in the presence of hydrogen peroxide, the oxidation of halide ions (Cl−, Br− or I−). They are classified according to the most electronegative halide that they can oxidize. Since the first discovery of a vanadium bromoperoxidase in the brown alga Ascophyllum nodosum thirty years ago, structural and mechanistic studies have been mainly conducted on two types of VHPO, chloro- and bromoperoxidases, and more recently on a vanadium-dependent iodoperoxidase. In this review, we highlight the main progress obtained on the structure-function relation of these proteins, based on biochemistry, crystallography and X-ray absorption spectroscopy (XAS). The comparison of 3D protein structures of the different VHPO helped identify the residues that govern the molecular mechanisms of catalysis and specificity of VHPO. Vanadium K-edge XAS gave further important insight to understand the fine changes around the vanadium cofactor during the catalytic cycle. The combination of different structural approaches, at different scales of resolution, shed new light on biological vanadium coordination in the active site, and its importance for the catalytic cycle and halide specificity of vanadium haloperoxidases.
[Show abstract][Hide abstract] ABSTRACT: The family 117 glycoside hydrolase (GH117) enzymes have exo-α-1,3-(3,6-anhydro)-L-galactosidase activity, removing terminal nonreducing α-1,3-linked 3,6-anhydro-L-galactose residues from their red algal neoagarose substrate. These enzymes have previously been phylogenetically divided into clades, and only the clade A enzymes have been experimentally studied to date. The investigation of two GH117 enzymes, Zg3615 and Zg3597, produced by the marine bacterium
reveals structural, biochemical and further phylogenetic diversity between clades. A product complex with the unusual β-3,6-anhydro-L-galactose residue sheds light on the inverting catalytic mechanism of the GH117 enzymes as well as the structure of this unique sugar produced by hydrolysis of the agarophyte red algal cell wall.
[Show abstract][Hide abstract] ABSTRACT: Laminarin is a β-1,3-D-glucan displaying occasional β-1,6 branches. This storage polysaccharide of brown algae constitutes an abundant source of carbon for marine bacteria such as Zobellia galactanivorans. This marine member of the Bacteroidetes possesses five putative β-1,3-glucanases [four belonging to glycosyl hydrolase family 16 (GH16) and one to GH64] with various modular architectures. Here, the characterization of the β-glucanase ZgLamC is reported. The catalytic GH16 module (ZgLamCGH16) was produced in Escherichia coli and purified. This recombinant enzyme has a preferential specificity for laminarin but also a significant activity on mixed-linked glucan (MLG). The structure of an inactive mutant of ZgLamCGH16 in complex with a thio-β-1,3-hexaglucan substrate unravelled a straight active-site cleft with three additional pockets flanking subsites -1, -2 and -3. These lateral pockets are occupied by a glycerol, an acetate ion and a chloride ion, respectively. The presence of these molecules in the vicinity of the O6 hydroxyl group of each glucose moiety suggests that ZgLamCGH16 accommodates branched laminarins as substrates. Altogether, ZgLamC is a secreted laminarinase that is likely to be involved in the initial step of degradation of branched laminarin, while the previously characterized ZgLamA efficiently degrades unbranched laminarin and oligo-laminarins.
[Show abstract][Hide abstract] ABSTRACT: Extreme ultraviolet photon activation tandem mass spectrometry (MS) at 69 nm (18 eV) was used to characterize mixtures of oligo-porphyrans, a class of highly sulfated oligosaccharides. Porphyrans, hybrid polymers whose structures are far from known, continue to provide a challenge for analytical method development. Activation by 18-eV photons led to a rich frag-mentation of the oligo-porphyrans, with many cross-ring and glycosidic cleavages. In contrast to multi-stage MSn strategies such as activated electron photodetachment dissociation, a single step of irradiation by energetic UV of multiply-charged anions led to a complete fragmentation of the oligo-porphyrans. In both ionization modes, the sulfate groups were retained on the backbone, which allowed the pattern of these modifications along the porphyran backbone to be described in unprecedented detail. Many structures released by the enzymatic degradation of the porphyran were completely resolved, including isomers. This work extends the existing knowledge on the structure of porphyrans. In addition, it provides a new demonstration of the potential of activation by high-energy photons for the structural analysis of oligosaccharides, even in unseparated mixtures, with a particular focus on sulfated compounds.
[Show abstract][Hide abstract] ABSTRACT: The eukaryotic initiation factor eIF4E is essential for cap-dependent initiation of translation in eukaryotes. Abnormal regulation of eIF4E has been implicated in oncogenic transformation. We developed an eIF4E-binding peptide derived from Angel1, a partner of eIF4E that we recently identified. We show here that this peptide fused to a penetratin motif causes drastic and rapid cell death in several epithelial cancer cell lines. This necrotic cell death was characterized by a drop in ATP levels with F-actin network injury being a key step in extensive plasma membrane blebbing and membrane permeabilization. This synthetic eIF4E-binding peptide provides a candidate pharmacophore for a promising new cancer therapy strategy.
[Show abstract][Hide abstract] ABSTRACT: At present, our molecular knowledge of dystrophin, the protein encoded by the DMD gene and mutated in myopathy patients, remains limited. To get around the absence of its atomic structure, we have developed an innovative interactive docking method based on the BioSpring software in combination with Small-angle X-ray Scattering (SAXS) data. BioSpring allows interactive handling of biological macromolecules thanks to an augmented Elastic Network Model (aENM) that combines the spring network with non-bonded terms between atoms or pseudo-atoms. This approach can be used for building molecular assemblies even on a desktop or a laptop computer thanks to code optimizations including parallel computing and GPU programming. By combining atomistic and coarse-grained models, the approach significantly simplifies the set-up of multi-scale scenarios. BioSpring is remarkably efficient for the preparation of numeric simulations or for the design of biomolecular models integrating qualitative experimental data restraints. The combination of this program and SAXS allowed us to propose the first high-resolution models of the filamentous central domain of dystrophin, covering repeats 11 to 17. Low-resolution interactive docking experiments driven by a potential grid enabled us to propose how dystrophin may associate with F-actin and nNOS. This information provides an insight into medically relevant discoveries to come.
[Show abstract][Hide abstract] ABSTRACT: Marine algae contribute approximately half of the global primary production. The large amounts of polysaccharides synthesized by these algae are degraded and consumed by microbes that utilize carbohydrate-active enzymes (CAZymes), thus creating one of the largest and most dynamic components of the Earth's carbon cycle. Over the last decade, structural and functional characterizations of marine CAZymes have revealed a diverse set of scaffolds and mechanisms that are used to degrade agars, carrageenan, alginate and ulvan-polysaccharides from red, brown and green seaweeds, respectively. The analysis of these CAZymes is not only expanding our understanding of their functions but is enabling the enhanced annotation of (meta)-genomic data sets, thus promoting an improved understanding of microbes that drive this marine component of the carbon cycle. Furthermore, this information is setting a foundation that will enable marine algae to be harnessed as a novel resource for biorefineries. In this review, we cover the most recent structural and functional analyses of marine CAZymes that are specialized in the digestion of macro-algal polysaccharides.
Current Opinion in Structural Biology 10/2014; 28(1):77–86. DOI:10.1016/j.sbi.2014.07.009 · 7.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vanadium haloperoxidases (VHPO) are key enzymes that oxidize halides and are involved in the biosynthesis of organo-halogens. Up to now only chloro-(VCPO) and bromoperoxidases (VBPO) have been structurally characterized, mainly from Eukaryotic species. Three putative VHPO genes were predicted in the genome of the flavobacterium Zobellia galactanivorans, a marine bacterium associated with macroalgae. In a phylogenetic analysis, these putative bacterial VHPO are closely related to other VHPO from diverse bacterial phyla, but cluster independently from eukaryotic algal VBPO and fungal VCPO. Two of these bacterial VHPO, heterogeneously produced in E. coli, were found to be strictly specific for iodide oxidation. The crystal structure of one of these vanadium-dependent iodoperoxidases, Zg-VIPO1, was solved by Multi-wavelength Anomalous Diffraction at 1.8 Å, revealing a monomeric structure mainly folded into α-helices. This 3D structure is relatively similar to those of VCPO of the fungus Curvularia inaequalis and of Streptomyces sp., and superimposable onto the dimeric structure of algal VBPO. Surprisingly, the vanadate binding site of Zg-VIPO1 is strictly conserved with the fungal VCPO active site. Using site-directed mutagenesis we showed that specific amino-acids and the associated hydrogen-bonding network around the vanadate center are essential for the catalytic properties and also for the iodide specificity of Zg-VIPO1. Altogether, phylogeny and structure-function data support the finding that iodoperoxidase activities evolved independently in bacterial and algal lineages, and this sheds light on the evolution of the VHPO enzyme family.
[Show abstract][Hide abstract] ABSTRACT: Ulvans are cell wall matrix polysaccharides in green algae belonging to the genus Ulva. Enzymatic degradation of the polysaccharide by ulvan lyases leads to the production of oligosaccharides with an unsaturated β-glucuronyl residue located at the non-reducing end. Exploration of the genomic environment around the Nonlabens ulvanivorans (previously Percicivirga ulvanivorans) ulvan lyase revealed a gene highly similar to known unsaturated uronyl hydrolases classified in the CAZy glycoside hydrolase family 105. The gene was cloned, the protein was overexpressed in E. coli, and enzymology experiments demonstrated its unsaturated β-glucuronyl activity. Kinetic analysis of purified oligo-ulvans incubated with the new enzyme showed that the full substrate specificity is attained by three subsites that preferentially bind anionic residues (sulfated rhamnose, glucuronic/iduronic acid). The 3D crystal structure of the native enzyme reveals that a trimeric organization is required for substrate binding and recognition at the +2 binding subsite. This novel unsaturated β-glucuronyl hydrolase is part of a previously uncharacterized subgroup of GH105 members and exhibits only a very limited sequence similarity to known unsaturated α-glucuronyl sequences previously found only in family GH88. Clan-O formed by families GH88 and GH105 was singular in the fact that it covered families acting on both axial and equatorial glycosidic linkages, respectively. The overall comparison of active site structures between enzymes from these two families highlights how that within family GH105, and unlike for classical glycoside hydrolysis, the hydrolysis of vinyl ether groups from unsaturated saccharides occurs independently of the α- or β-configuration of the cleaved linkage.
[Show abstract][Hide abstract] ABSTRACT: Laminarinase is commonly used to describe [beta]-1,3-glucanases widespread throughout Archaea, bacteria and several eukaryotic lineages. Some [beta]-1,3-glucanases have already been structurally and biochemically characterized, but very few from organisms that are in contact with genuine laminarin, the storage polysaccharide of brown algae. Here we report the heterologous expression and subsequent biochemical and structural characterization of ZgLamAGH16 from Zobellia galactanivorans, the first GH16 laminarinase from a marine bacterium associated with seaweeds. ZgLamAGH16 contains a unique additional loop, compared to other GH16 laminarinases, which is composed of 17 amino-acids and gives a bent shape to the active cleft of the enzyme. This particular topology is perfectly adapted to the U-shape conformation of laminarin chains in solution, and thus explains the predominant specificity of ZgLamAGH16 for this substrate. The 3D structure of the enzyme and two enzyme-substrate complexes, one with laminaritetraose, the other with a trisaccharide of 1,3-1,4-[beta]-D-glucan, have been determined at 1.5 Å, 1.35 Å and 1.13 Å resolution, respectively. The structural comparison of substrate recognition pattern between these complexes allow the proposition that ZgLamAGH16 likely diverged from an ancestral broad specificity GH16 β-glucanase and evolved toward a bent active site topology adapted to efficient degradation of algal laminarin.
[Show abstract][Hide abstract] ABSTRACT: A large number of retaining glycosidases catalyze both hydrolysis and transglycosylation reactions, but little is known about what determines the balance between these two activities (transglycosylation/hydrolysis ratio). We previously obtained by directed evolution the mutants F401S and N282T of Thermus thermophilus β-glycosidase (Ttβ-gly, glycoside hydrolase family 1 (GH1)), which display a higher transglycosylation/hydrolysis ratio than the wild-type enzyme. In order to find the cause of these activity modifications, and thereby set up a generic method for easily obtaining transglycosidases from glycosidases, we determined their X-ray structure. No major structural changes could be observed which could help to rationalize the mutagenesis of glycosidases into transglycosidases. However, as these mutations are highly conserved in GH1 β-glycosidases and are located around the -1 site, we pursued the isolation of new transglycosidases by targeting highly conserved amino acids located around the active site. Thus, by single-point mutagenesis on Ttβ-gly, we created four new mutants that exhibit improved synthetic activity, producing disaccharides in yields of 68-90% against only 36% when native Ttβ-gly was used. As all of the chosen positions were well conserved among GH1 enzymes, this approach is most probably a general route to convert GH1 glycosidases into transglycosidases.
Protein Engineering Design and Selection 11/2013; 27(1). DOI:10.1093/protein/gzt057 · 2.54 Impact Factor