[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: 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; · 8.28 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: Zobellia galactanivorans is an emerging model bacterium for the bioconversion of algal biomass. Notably, this marine Bacteroidetes possesses a complex agarolytic system comprising four β-agarases and five β-porphyranases, all belonging to the glycoside hydrolase family 16. Although β-agarases are specific for the neutral agarobiose moieties, the recently discovered β-porphyranases degrade the sulfated polymers found in various quantities in natural agars. Here, we report the biochemical and structural comparison of five β-porphyranases and β-agarases from Z. galactanivorans. The respective degradation patterns of two β-porphyranases and three β-agarases are analyzed by their action on defined hybrid oligosaccharides. In light of the high resolution crystal structures, the biochemical results allowed a detailed mapping of substrate specificities along the active site groove of the enzymes. Although PorA displays a strict requirement for C6-sulfate in the -2- and +1-binding subsites, PorB tolerates the presence of 3-6-anhydro-l-galactose in subsite -2. Both enzymes do not accept methylation of the galactose unit in the -1 subsite. The β-agarase AgaD requires at least four consecutive agarose units (DP8) and is highly intolerant to modifications, whereas for AgaB oligosaccharides containing C6-sulfate groups at the -4, +1, and +3 positions are still degraded. Together with a transcriptional analysis of the expression of these enzymes, the structural and biochemical results allow proposition of a model scheme for the agarolytic system of Z. galactanivorans.
Journal of Biological Chemistry 07/2012; 287(36):30571-84. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Signaling across the membrane in response to extracellular stimuli is essential for survival of all cells. In bacteria, responses to environmental changes are predominantly mediated by two-component systems, which are typically composed of a membrane-spanning sensor histidine kinase and a cytoplasmic response regulator. In the human gut symbiont Bacteroides thetaiotaomicron, hybrid two-component systems are a key part of the bacterium's ability to sense and degrade complex carbohydrates in the gut. Here, we identify the activating ligand of the hybrid two-component system, BT4663, which controls heparin and heparan sulfate acquisition and degradation in this prominent gut microbe, and report the crystal structure of the extracellular sensor domain in both apo and ligand-bound forms. Current models for signal transduction across the membrane involve either a piston-like or rotational displacement of the transmembrane helices to modulate activity of the linked cytoplasmic kinases. The structures of the BT4663 sensor domain reveal a significant conformational change in the homodimer on ligand binding, which results in a scissor-like closing of the C-termini of each protomer. We propose this movement activates the attached intracellular kinase domains and represents an allosteric mechanism for bacterial transmembrane signaling distinct from previously described models, thus expanding our understanding of signal transduction across the membrane, a fundamental requirement in many important biological processes.
Proceedings of the National Academy of Sciences 04/2012; 109(19):7298-303. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Alginate constitutes a significant part of seaweed biomass and thus a crucial nutrient for numerous marine heterotrophic bacteria. However, the mechanisms for alginate assimilation remain largely unknown in marine microorganisms. We show here that the genome of the marine flavobacterium Zobellia galactanivorans contains seven putative alginate lyase genes, five of them localized within two clusters comprising additional carbohydrate-related genes. The transcription of these genes and the alginolytic activity were strongly induced when Z. galactanivorans used alginate as sole carbon source. These clusters were shown to be transcribed as polycistronic mRNAs and thus to constitute operons. Several candidate enzymes were successfully overexpressed in Escherichia coli, purified and their activity tested. Particularly, AlyA1, AlyA4, AlyA5 and AlyA7 are confirmed as active alginate lyases. Zg2622 and Zg2614 are a dehydrogenase and a kinase, respectively, further converting the terminal unsaturated monosaccharides released by alginate lyases into 2-keto-3-deoxy-6-phosphogluconate. In-depth phylogenomic analyses reveal that such alginolytic operons originated from an ancestral marine flavobacterium and were independently transferred to marine proteobacteria and Japanese gut Bacteroides. These bacteria thus gained the capacity to assimilate the main polysaccharide of brown algae, an adaptive advantage in coastal environments but also in the gut microbiota of specific human population.
[show abstract][hide abstract] ABSTRACT: The gene coding for an alginate lyase from the marine bacteria Pseudomonas alginovora X017 was cloned and heterologously expressed in Escherichia coli strains. The protein was produced in inclusion bodies and the active form was obtained by applying a refolding protocol based upon dilution. The biochemical characterization was performed on the active, refolded form of the alginate lyase. The substrate specificity was monitored by NMR. The degradation products were size-fractioned by size exclusion chromatography. The fractions were subsequently analyzed by ESI-MS to determine the molecular weight of the compounds. The structures of the different oligosaccharides were then elucidated by NMR. The enzyme was shown to be only acting on M-M diads. No enzymatic hydrolysis occurred between M-MG, G-MM or G-MG blocks proving that the sequence accounting for the generated oligomers by enzymatic hydrolysis is M-MM. The unsaturated oligosaccharides produced by the alginate lyase were ΔM, ΔMM, ΔMMM, and ΔMMMM indicating that the minimum structure recognized by the enzyme is the M6 oligosaccharide.
Carbohydrate research 02/2012; 352:44-50. · 2.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Small-angle X-ray scattering (SAXS) is an increasingly popular method to obtain low-resolution structures of complex macromolecules and their complexes in solution, in part due to recent technical and computational advances that make this method more and more accessible. However, to obtain unambiguous molecular interpretation from SAXS envelopes, the efficient use of and combination with additional structural methods are crucial. The multimodular character of cellulases and their assemblage in the cellulosome are ideally analyzed by such a combination of structural methods. Here, we describe how information from different sources can be combined with SAXS to determine the molecular organization and we depict the recent advancements and trends that are leading to a more comprehensive picture of the molecular architecture of these multimodular enzymes and their organization in macro-assemblages such as cellulosomes.
Methods in enzymology 01/2012; 510:183-210. · 1.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: SpuA is a large multimodular cell wall-attached enzyme involved in the degradation of glycogen by the pathogenic bacterium Streptococcus pneumoniae. The deletion of the gene encoding SpuA from the bacterium resulted in a strain with reduced competitiveness in a mouse model of virulence relative to the parent strain, linking the degradation of host-glycogen to the virulence of the bacterium. Through the combined use of X-ray crystallography, small-angle X-ray scattering, and inhibitor binding, the molecular features involved in substrate recognition by this complex protein are revealed. This uniquely illustrates the complexity of the active site, the conformational changes incurred during carbohydrate binding by this protein, and the interaction and cooperation of its composite modules during this process. New insight into the function of this particular pneumococcal virulence factor is provided along with substantial contributions to the nascent framework for understanding the structural and functional interplay between modules in multimodular carbohydrate-active enzymes.
[show abstract][hide abstract] ABSTRACT: The genomic data on heterotrophic marine bacteria suggest the crucial role that microbes play in the global carbon cycle. However, the massive presence of hypothetical proteins hampers our understanding of the mechanisms by which this carbon cycle is carried out. Moreover, genomic data from marine microorganisms are essentially annotated in the light of the biochemical knowledge accumulated on bacteria and fungi which decompose terrestrial plants. However marine algal polysaccharides clearly differ from their terrestrial counterparts, and their associated enzymes usually constitute novel protein families. In this study, we have applied a combination of bioinformatics, targeted activity screening and structural biology to characterize a hypothetical protein from the marine bacterium Zobellia galactanivorans, which is distantly related to GH43 family. This protein is in fact a 1,3-α-3,6-anhydro-l-galactosidase (AhgA) which catalyses the last step in the degradation pathway of agars, a family of polysaccharides unique to red macroalgae. AhgA adopts a β-propeller fold and displays a zinc-dependent catalytic machinery. This enzyme is the first representative of a new family of glycoside hydrolases, especially abundant in coastal waters. Such genes of marine origin have been transferred to symbiotic microbes associated with marine fishes, but also with some specific human populations.
[show abstract][hide abstract] ABSTRACT: Molecular dynamics simulations of the tetradecasaccharide XXXGXXXG in complex with the hybrid aspen xyloglucan endo-transglycosylase PttXET16-34 have been performed and analysed with respect to structure, dynamics, flexibility and ligand interactions. Notably, the charge state of the so-called ‘helper residue’ aspartate 87 (Asp87), which lies between the catalytic nucleophile [glutamate 85 (Glu85)] and general acid/base (Glu89) residues on the same beta strand, had a significant effect on PttXET16-34 active site structure. When Asp87 was deprotonated, electrostatic repulsion forced the nucleophile away from C1 of the sugar ring in subsite − 1 and the proton–donating ability of Glu89 was also weakened due to the formation of a hydrogen bond with Asp87, whereas the protonation of Asp87 resulted in the formation of a hydrogen bond with the catalytic nucleophile and correct positioning of the catalytic machinery. The results suggest that catalysis in glycoside hydrolase family 16, and by extension clan GH-B enzymes, is optimal when the catalytic nucleophile is deprotonated for nucleophilic attack on the substrate, whereas the ‘helper residue’ and general acid/base residue are both in their conjugate acid forms to align the nucleophile and deliver a proton to the departing sugar, respectively.
[show abstract][hide abstract] ABSTRACT: The transforming growth factor (TGF)-β superfamily is a group of important growth factors involved in multiple processes such as differentiation, cell proliferation, apoptosis and cellular growth. In the Pacific oyster Crassostrea gigas, the oyster gonadal (og) TGF-β gene was recently characterized through genome-wide expression profiling of oyster lines selected to be resistant or susceptible to summer mortality. Og TGF-β appeared specifically expressed in the gonad to reach a maximum when gonads are fully mature, which singularly contrasts with the pleiotropic roles commonly ascribed to most TGF-β family members. The function of og TGF-β protein in oysters is unknown, and defining its role remains challenging. In this study, we develop a rapid bacterial production system to obtain recombinant og TGF-β protein, and we demonstrate that og TGF-β is processed by furin to a mature form of the protein. This mature form can be detected in vivo in the gonad. Functional inhibition of mature og TGF-β in the gonad was conducted by inactivation of the protein using injection of antibodies. We show that inhibition of og TGF-β function tends to reduce gonadic area. We conclude that mature og TGF-β probably functions as an activator of germ cells development in oyster.
[show abstract][hide abstract] ABSTRACT: Members of the diverse bacterial phylum Bacteroidetes have colonized virtually all types of habitats on Earth. They are among the major members of the microbiota of animals, especially in the gastrointestinal tract, can act as pathogens and are frequently found in soils, oceans and freshwater. In these contrasting ecological niches, Bacteroidetes are increasingly regarded as specialists for the degradation of high molecular weight organic matter, i.e., proteins and carbohydrates. This review presents the current knowledge on the role and mechanisms of polysaccharide degradation by Bacteroidetes in their respective habitats. The recent sequencing of Bacteroidetes genomes confirms the presence of numerous carbohydrate-active enzymes covering a large spectrum of substrates from plant, algal, and animal origin. Comparative genomics reveal specific Polysaccharide Utilization Loci shared between distantly related members of the phylum, either in environmental or gut-associated species. Moreover, Bacteroidetes genomes appear to be highly plastic and frequently reorganized through genetic rearrangements, gene duplications and lateral gene transfers (LGT), a feature that could have driven their adaptation to distinct ecological niches. Evidence is accumulating that the nature of the diet shapes the composition of the intestinal microbiota. We address the potential links between gut and environmental bacteria through food consumption. LGT can provide gut bacteria with original sets of utensils to degrade otherwise refractory substrates found in the diet. A more complete understanding of the genetic gateways between food-associated environmental species and intestinal microbial communities sheds new light on the origin and evolution of Bacteroidetes as animals' symbionts. It also raises the question as to how the consumption of increasingly hygienic and processed food deprives our microbiota from useful environmental genes and possibly affects our health.
[show abstract][hide abstract] ABSTRACT: Porphyran from the red seaweed Porphyra umbilicalis was degraded with β-porphyranase A derived from the marine flavobacterium Zobellia galactanivorans. β-Porphyranase A produces a new series of sulfated oligosaccharides that we characterized by NMR, HPAEC and size-exclusion chromatography. In contrast to previously used β-agarases, which produce predominantly non-sulfated oligosaccharides of the neoagarobiose series and hybrid oligosaccharides, β-porphyranase A produced oligosaccharides of the α-l-Galp-6-sulfate (1 → 3) ß-d-Galp (L6S-G) series and the L6S-G disaccharide was the major final product. The newly identified β-porphyranase A was further used in combination with β-agarase B to revise porphyran polysaccharide structure.
[show abstract][hide abstract] ABSTRACT: eIF4E binding protein (4E-BP) inhibits translation of capped mRNA by binding to the initiation factor eIF4E and is known to be mostly or completely unstructured in both free and bound states. Using small angle X-ray scattering (SAXS), we report here the analysis of 4E-BP structure in solution, which reveals that while 4E-BP is intrinsically disordered in the free state, it undergoes a dramatic compaction in the bound state. Our results demonstrate that 4E-BP and eIF4E form a 'fuzzy complex', challenging current visions of eIF4E/4E-BP complex regulation.
Nucleic Acids Research 12/2010; 39(8):3496-503. · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: Marine polysaccharide degrading enzymes, and iota-carrageenases in particular, have received little attention in the past, although their substrate specificity is of interest for biotechnological applications. This is mostly a consequence of the lack of data about their occurrence in the marine environment. Recent metagenomic data mining and the genome sequencing of a marine bacterium, Zobellia galactanivorans, led to the identification of three new iota-carrageenase genes belonging to the glycoside hydrolase family GH82. The additional sequences helped to identify potential candidate residues as catalytic proton donor and nucleophile. We have identified the catalytic key residues experimentally by site-directed mutagenesis and subsequent kinetic analysis for the iota-carrageenase from Alteromonas fortis CgiA1_Af. The kinetic analyses of the purified mutant enzymes confirm that E245 plays the role of the catalytic proton donor and D247 the general base that activates the catalytic water molecule. The point mutations of three other residues, namely, Q222, H281, and Q310 in A. fortis, located in proximity of the active site also affect the enzyme activity. Our results indicate that E310 plays a role in stabilizing the substrate intermediate conformation, while H281 is involved in substrate binding and appears to be crucial for maintaining the protonation state of the catalytic proton donor E245. The third residue, Q222, that bridges the catalytic water molecule and a chloride ion, plays a crucial role in structuring the water network in the active site of A. fortis iota-carrageenase.
[show abstract][hide abstract] ABSTRACT: Marine bacteria secrete specific glycoside hydrolases such as agarases to access polysaccharides from algal cell walls as a carbon and energy source. In an attempt to identify agarases with variable degradation patterns, a novel family GH16 beta-agarase from the marine bacterium Zobellia galactanivorans was expressed, purified and crystallized. The purified enzyme crystallized in two distinct forms that were grown by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. Hexagonal crystals belonging to space group P3(1)21 diffracted to 2.2 A resolution, whereas orthorhombic crystals belonging to space group P2(1)2(1)2(1) diffracted to 1.5 A resolution.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 04/2010; 66(Pt 4):413-7. · 0.55 Impact Factor