[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.
Applied and Environmental Microbiology 09/2014; · 3.95 Impact Factor
[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.
Journal of Biological Chemistry 01/2014; · 4.65 Impact Factor
[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 01/2014; 28:77–86. · 8.74 Impact Factor
[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.
Journal of Biological Chemistry 12/2013; · 4.65 Impact Factor
[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; · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Marine bacteria are precious sources for the isolation of enzymes specializing in brown, red and green macro-algal polysaccharide degradation. The marine enzyme discovery has basically been performed by the isolation and purification of defined activities, leading to an evident bias driven by those that are industrially produced, and consequently to a lack of enzymes active on more scarcely distributed marine polysaccharides. Interestingly, the discovery of the first bacterial enzymes active on algal polysaccharides revealed that many of these displayed only very distant structural similarity to known terrestrial polysaccharide degrading enzymes, and more often belonged to new glycoside hydrolase families. In this chapter, we review the general properties of polysaccharide-degrading enzymes originating from marine bacteria, giving a special focus on agarases and carrageenases, but also including alginate lyases, fucanolytic enzymes and ulvan degrading enzymes. The chapter concludes with an outlook towards potential applications and the need for more global approaches to deal with modern biotechnological requirements.
[Show abstract][Hide abstract] ABSTRACT: Brown algal phlorotannins are structural analogs of condensed tannins in terrestrial plants and like plant phenols, they have numerous biological functions. Despite their importance in brown algae, phlorotannin biosynthetic pathways have been poorly characterized at the molecular level. We found that a predicted type III polyketide synthase in the genome of the brown alga Ectocarpus siliculosus, PKS1, catalyzes a major step in the biosynthetic pathway of phlorotannins (i.e., the synthesis of phloroglucinol monomers from malonyl-CoA). The crystal structure of PKS1 at 2.85-Å resolution provided a good quality electron density map showing a modified Cys residue, likely connected to a long chain acyl group. An additional pocket not found in other known type III PKSs contains a reaction product that might correspond to a phloroglucinol precursor. In vivo, we also found a positive correlation between the phloroglucinol content and the PKS III gene expression level in cells of a strain of Ectocarpus adapted to freshwater during its reacclimation to seawater. The evolution of the type III PKS gene family in Stramenopiles suggests a lateral gene transfer event from an actinobacterium.
[Show abstract][Hide abstract] ABSTRACT: The initiation factor 4E (eIF4E) is implicated in most of the crucial steps of the mRNA life cycle and is recognized as a pivotal protein in gene regulation. Many of these roles are mediated by its interaction with specific proteins generally known as eIF4E-interacting partners (4E-IPs), such as eIF4G and 4E-BP. To screen for new 4E-IPs, we developed a novel approach based on structural, in silico and biochemical analyses. We identified the protein Angel1, a member of the CCR4 deadenylase family. Immunoprecipitation experiments provided evidence that Angel1 is able to interact in vitro and in vivo with eIF4E. Point mutation variants of Angel1 demonstrated that the interaction of Angel1 with eIF4E is mediated through a consensus eIF4E-binding motif. Immunofluorescence and cell fractionation experiments showed that Angel1 is confined to the endoplasmic reticulum and Golgi apparatus, where it partially co-localizes with eIF4E and eIF4G, but not with 4E-BP. Furthermore, manipulating Angel1 levels in living cells had no effect on global translation rates, suggesting that the protein has a more specific function. Taken together, our results illustrate that we developed a powerful method for identifying new eIF4E partners and open new perspectives for understanding eIF4E-specific regulation.
Nucleic Acids Research 06/2013; · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cell walls of brown algae are complex supramolecular assemblies containing various original, sulfated and carboxylated polysaccharides. Among these, the major marine polysaccharide component, alginate, represents an important biomass that is successfully turned over by heterotrophic marine bacteria. In the marine flavobacterium Zobellia galactanivorans the catabolism and uptake of alginate is encoded by operon structures that resemble the typical Bacteroidetes polysaccharide utilization locus (PUL). The genome of Z. galactanivorans contains seven putative alginate lyase genes, five of which are localized within two clusters comprising additional carbohydrate-related genes. This study reports the detailed biochemical and structural characterization of two of these. We demonstrate here that AlyA1PL7 is an endolytic guluronate lyase, while AlyA5 cleaves unsaturated units, a-L-guluronate (G) or b-D-manuronate (M) residues at the non-reducing end of oligo-alginates in an exolytic fashion. Despite a common jelly-roll fold, these striking differences of mode of action are explained by a distinct active site topology: an open cleft in AlyA1PL7, whereas AlyA5 displays a pocket topology due to the presence of additional loops partially obstructing the catalytic groove. Finally, in contrast to PL7 alginate lyases from terrestrial bacteria, both enzymes proceed according a calcium-dependent mechanism suggesting an exquisite adaptation to their natural substrate in the context of brown algal cell walls.
Journal of Biological Chemistry 06/2013; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Red seaweeds are key components of coastal ecosystems and are economically important as food and as a source of gelling agents, but their genes and genomes have received little attention. Here we report the sequencing of the 105-Mbp genome of the florideophyte Chondrus crispus (Irish moss) and the annotation of the 9,606 genes. The genome features an unusual structure characterized by gene-dense regions surrounded by repeat-rich regions dominated by transposable elements. Despite its fairly large size, this genome shows features typical of compact genomes, e.g., on average only 0.3 introns per gene, short introns, low median distance between genes, small gene families, and no indication of large-scale genome duplication. The genome also gives insights into the metabolism of marine red algae and adaptations to the marine environment, including genes related to halogen metabolism, oxylipins, and multicellularity (microRNA processing and transcription factors). Particularly interesting are features related to carbohydrate metabolism, which include a minimalistic gene set for starch biosynthesis, the presence of cellulose synthases acquired before the primary endosymbiosis showing the polyphyly of cellulose synthesis in Archaeplastida, and cellulases absent in terrestrial plants as well as the occurrence of a mannosylglycerate synthase potentially originating from a marine bacterium. To explain the observations on genome structure and gene content, we propose an evolutionary scenario involving an ancestral red alga that was driven by early ecological forces to lose genes, introns, and intergenetic DNA; this loss was followed by an expansion of genome size as a consequence of activity of transposable elements.
Proceedings of the National Academy of Sciences 03/2013; 110(13):5247-5252. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Neuroglobins, previously thought to be restricted to vertebrate neurons, were detected in the brain of a photosymbiotic acoel, Symsagittifera roscoffensis, and in neuro-sensory cells of the jellyfish Clytia hemisphaerica. For the neuroglobin of S. roscoffensis, a member of a lineage that originated either at the base of the bilateria or of the deuterostome clade, we report the ligand-binding properties, crystal structure at 2.3 A and brain immuno-cytochemical pattern. We also describe in situ hybridizations of two neuroglobins specifically expressed in differentiating nematocytes (neuro-sensory cells) and in statocytes (ciliated mechano-sensory cells) of C. hemisphaerica, a member of the early-branching animal phylum cnidaria. In silico searches using these neuroglobins as queries revealed the presence of previously unidentified neuroglobin-like sequences in most metazoan lineages. Since neural systems are almost ubiquitous in metazoa, the constitutive expression of neuroglobin-like proteins strongly supports the notion of an intimate association of neuroglobins with the evolution of animal neural systems and hints at the preservation of a vitally important function. Neuroglobins were probably recruited in the first proto-neurons in early metazoans from globin precursors. Neuroglobins were identified in choanoflagellates, sponges and placozoans, and were conserved during nervous system evolution. Since the origin of neuroglobins predates the other metazoan globins, it is likely that neuroglobin gene duplication followed by co-option and subfunctionalization led to the emergence of globin families in protostomes and deuterostomes (i.e convergent evolution).
Journal of Biological Chemistry 01/2013; · 4.65 Impact Factor