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CAZypedia was initiated in 2007 to create a comprehensive, living encyclopedia of the carbohydrate-active enzymes (CAZymes) and associated carbohydrate-binding modules involved in the synthesis, modification, and degradation of complex carbohydrates. CAZypedia is closely connected with the actively-curated CAZy database, which provides a sequence-based foundation for the biochemical, mechanistic, and structural characterization of these diverse proteins. Now celebrating its 10th anniversary online, CAZypedia is a successful example of dynamic, community-driven, and expert-based biocuration. CAZypedia is an open-access resource available at URL http://www.cazypedia.org.

Masitinib, a highly selective protein kinase inhibitor, can sensitise gemcitabine-refractory cancer cell lines when used in combination with gemcitabine. Here we report a reverse proteomic approach that identifies the target responsible for this sensitisation: the deoxycytidine kinase (dCK). Masitinib, as well as other protein kinase inhibitors, such as imatinib, interact with dCK and provoke an unforeseen conformational-dependent activation of this nucleoside kinase, modulating phosphorylation of nucleoside analogue drugs. This phenomenon leads to an increase of prodrug phosphorylation of most of the chemotherapeutic drugs activated by this nucleoside kinase. The unforeseen dual activity of protein kinase inhibition/nucleoside kinase activation could be of great therapeutic benefit, through either reducing toxicity of therapeutic agents by maintaining effectiveness at lower doses or by counteracting drug resistance initiated via down modulation of dCK target., Masitinib is a protein kinase inhibitor that sensitises refractory pancreatic adenocarcinoma cells to treatment with the nucleoside analog gemcitabine. Here the authors show that Masitinib activates deoxycytidine kinase to enhance phosphorylation of nucleoside analogue pro-drugs, increasing their potency.

Vanadium haloperoxidases (VHPO) are key enzymes that oxidize halides and are involved in the biosynthesis of organo-halogens. Until now, only chloroperoxidases (VCPO) and bromoperoxidases (VBPO) have been characterized structurally, 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 were closely related to other VHPO from diverse bacterial phyla but clustered independently from eukaryotic algal VBPO and fungal VCPO. Two of these bacterial VHPO, heterogeneously produced in Escherichia 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 multiwavelength anomalous diffraction at 1.8 Å, revealing a monomeric structure mainly folded into α-helices. This three-dimensional structure is relatively similar to those of VCPO of the fungus Curvularia inaequalis and of Streptomyces sp. and is 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 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.

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.

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.

A midthroughput screening follow-up program targeting the first bromodomain of the human BRD4 protein, BRD4(BD1), identified an acetylated-mimic xanthine derivative inhibitor. This compound binds with an affinity in the low micromolar range yet exerts suitable unexpected selectivity in vitro against the other members of the bromodomain and extra-terminal domain (BET) family. A structure-based program pinpointed a role of the ZA loop, paving the way for the development of potent and selective BET-BRDi probes.

Protein-protein interactions (PPIs) represent an enormous source of opportunity for therapeutic intervention. We and others have recently pinpointed key rules that will help in identifying the next generation of innovative drugs to tackle this challenging class of targets within the next decade. We used these rules to design an oriented chemical library corresponding to a set of diverse 'PPI-like' modulators with cores identified as privileged structures in therapeutics. In this work, we purchased the resulting 1664 structurally diverse compounds and evaluated them on a series of representative protein-protein interfaces with distinct "druggability" potential using Homogeneous Time-Resolved Fluorescence (HTRF®) technology. For certain PPI classes, analysis of the hit rates revealed up to 100 enrichment factors compared with non-oriented chemical libraries. This observation correlates with the predicted "druggability" of the targets. A specific focus on selectivity profiles, the three-dimensional (3D) molecular modes of action resolved by X-ray crystallography, and the biological activities of identified hits targeting the well-defined "druggable" bromodomains of the bromo and extraterminal (BET) family are presented as a proof-of-concept. Overall, our present study illustrates the potency of machine learning-based oriented chemical libraries to accelerate the identification of hits targeting PPIs. A generalization of this method to a larger set of compounds will accelerate the discovery of original and potent probes for this challenging class of targets.

International audience; 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.

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 bacteriumZobellia galactanivoransreveals 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.

Macromolecular crystallography is the most direct and accurate approach to determine the three-dimensional structure of biological macromolecules. The growth of high quality single crystals, yielding diffraction to the highest X-ray resolution, remains a bottleneck in this methodology. Here we show that through a modification of the batch crystallization method, an entirely convection-free crystallization environment is achieved, which enhances the purity and crystallinity of protein crystals. This is accomplished by using an upside-down geometry, where crystals grow at the “ceiling” of a growth-cell completely filled with the crystallization solution. The “ceiling crystals” experience the same diffusion-limited conditions as in space microgravity experiments. The new method was tested on bovine insulin and two hen egg-white lysozyme polymorphs. In all cases, ceiling crystals diffracted X-rays to resolution limits beyond that for other methods using similar crystallization conditions without further optim...

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.

Background The production of stable and soluble proteins is one of the most important steps prior to structural and functional studies of biological importance. We investigated the parallel production in a medium throughput strategy of genes coding for proteins from various marine organisms, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. This strategy was applied in order to respond to the need for post-genomic validation of the recent success of a large number of marine genomic projects. Indeed, the upcoming challenge is to go beyond the bioinformatic data, since the bias introduced through the genomes of the so called model organisms leads to numerous proteins of unknown function in the still unexplored world of the oceanic organisms. Results We present here the results of expression tests for 192 targets using a 96-well plate format. Genes were PCR amplified and cloned in parallel into expression vectors pFO4 and pGEX-4T-1, in order to express proteins N-terminally fused to a six-histidine-tag and to a GST-tag, respectively. Small-scale expression and purification permitted isolation of 84 soluble proteins and 34 insoluble proteins, which could also be used in refolding assays. Selected examples of proteins expressed and purified to a larger scale are presented. Conclusions The objective of this program was to get around the bottlenecks of soluble, active protein expression and crystallization for post-genomic validation of a number of proteins that come from various marine organisms. Multiplying the constructions, vectors and targets treated in parallel is important for the success of a medium throughput strategy and considerably increases the chances to get rapid access to pure and soluble protein samples, needed for the subsequent biochemical characterizations. Our set up of a medium throughput strategy applied to genes from marine organisms had a mean success rate of 44% soluble protein expression from marine bacteria, archaea as well as eukaryotic organisms. This success rate compares favorably with other protein screening projects, particularly for eukaryotic proteins. Several purified targets have already formed the base for experiments aimed at post-genomic validation.

The purple pigmented bacterium Chromobacterium violaceum is a dominant component of tropical soil microbiota that can cause rare but fatal septicaemia in humans. Its sequenced genome provides insight into the abundant potential of this organism for biotechnological and pharmaceutical applications and allowed an ORF encoding a protein that is 60% identical to the fucose binding lectin (PA-IIL) from Pseudomonas aeruginosa and the mannose binding lectin (RS-IIL) from Ralstonia solanacearum to be identified. The lectin, CV-IIL, has recently been purified from C. violaceum [Zinger-Yosovich, K., Sudakevitz, D., Imberty, A., Garber, N. C., and Gilboa-Garber, N. (2006) Microbiology 152, 457-463] and has been confirmed to be a tetramer with subunit size of 11.86 kDa and a binding preference for fucose. We describe here the cloning of CV-IIL and its expression as a recombinant protein. A complete structure-function characterization has been made in an effort to analyze the specificity and affinity of CV-IIL for fucose and mannose. Crystal structures of CV-IIL complexes with monosaccharides have yielded the molecular basis of the specificity. Each monomer contains two close calcium cations that mediate the binding of the monosaccharides, which occurs in different orientations for fucose and mannose. The thermodynamics of binding has been analyzed by titration microcalorimetry, giving dissociation constants of 1.7 and 19 microM for alpha-methyl fucoside and alpha-methyl mannoside, respectively. Further analysis demonstrated a strongly favorable entropy term that is unusual in carbohydrate binding. A comparison with both PA-IIL and RS-IIL, which have binding preferences for fucose and mannose, respectively, yielded insights into the monosaccharide specificity of this important class of soluble bacterial lectins.