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International audience; 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 surface of algae is colonized by marine heterotrophic bacteria which entertain different trophic relationships with their host. These relationships may be of various types such as symbiosis, commensalism, saprophytic or pathogenic. In all cases the algal cell wall, which is mainly constituted by polysaccharides, is the first barrier bacteria have to interact with. Therefore, bacteria have developed sophisticated machineries (involving various glycoside hydrolases) to use the cell wall components as carbon source, but they also have to cope with algal defence reactions, which are also located in the cell wall of algae. Zobellia galactanivorans is a marine flavobacteria whose genome has recently been sequenced. It represents a model for the interaction between algae and bacteria. Indeed, 3 % of genes are involved in polysaccharide degradation, many of them organised in operon-like structures. Moreover, many enzymes present in the genome appear to be involved in original metabolisms, such as iodine utilization. My interest focused on the molecular aspects of three different factors involved in interaction of Z. galactanivorans with algae. The genome of Z. galactanivorans revealed two new sequences of glycosides hydrolases family 82 which are particular, due to their truncated sequences. Based on multiple sequence alignments, I designed site directed mutagenesis experiments, performed with the Alteromonas fortis enzyme, the first structural representant of iota-carrageenases. The results show that E245 (proton donor) and D247 (nucleophile) are the catalytic residues and that residues Q222, H281 and E310 also play crucial roles in the enzymatic reaction. Using a crystallographic approach, I was able to highlight the importance of a chloride ion in the formation of a water network, close to the active site. I also initiated the first biochemical and structural characterization of two members of a new GH family, located in operon-like gene organisations apparently involved in the degradation of sulphated polysaccharides. A third part of my thesis concerns the crystallographic structure determination of a first prokaryote, vanadium dependent iodoperoxydase, identified in the Z. galactanivorans genome. The structural analysis of this enzyme that may be involved in a detoxification reaction following the oxidative burst generated by algae, allows us to suggest a new evolution pattern for this type of enzymes.; La surface des algues est colonisée par des bactéries marines hétérotrophes qui entretiennent avec leurs hôtes différentes relations trophiques. Zobellia galactanivorans, une flavobactérie marine, est un modèle de l'interaction entre les bactéries et les algues. 3 % des gènes de cette bactérie sont impliquées dans la dégradation de polysaccharides. De plus on retrouve, dans son génome, des enzymes participant à des métabolismes originaux comme celui de l'iode. Je me suis intéressé à 3 aspects moléculaires de l'interaction entre Z. galactanivorans et les algues. Premièrement le génome de Z. galactanivorans possède deux nouvelles iota-carraghénases de la famille GH 82, plus courtes que celles déjà décrites. Dans le but d'identifier la machinerie catalytique de ces enzymes, je me suis basé sur l'alignement multiple de l'ensemble des séquences de la famille GH 82 pour réaliser des expériences de mutagenèse dirigées sur la iotases d'Alteromonas fortis et ainsi expliquer le mécanisme catalytique de cette famille de glycoside hydrolase. J'ai également effectué une première caractérisation biochimique et structurale de deux membres d'une nouvelle famille de glycoside hydrolases, identifié, dans le génome de Z. galactanivorans, au sein d'opérons putatifs très probablement impliqués dans la dégradation de polysaccharides sulfatés. Enfin, j'ai résolu la première structure cristallographique d'une iodoperoxydase vanadium-dépendante d'origine bactérienne. Cette enzyme pourrait être impliquée dans un mécanisme de détoxification de la bactérie, suite à un ''burst oxydant'' généré par l'algue. Cette première structure permet de revoir et de discuter l'évolution de ces protéines.

The surface of algae is colonized by marine heterotrophic bacteria which entertain different trophic relationships with their host. These relationships may be of various types such as symbiosis, commensalism, saprophytic or pathogenic. In all cases the algal cell wall, which is mainly constituted by polysaccharides, is the first barrier bacteria have to interact with. Therefore, bacteria have developed sophisticated machineries (involving various glycoside hydrolases) to use the cell wall components as carbon source, but they also have to cope with algal defence reactions, which are also located in the cell wall of algae. Zobellia galactanivorans is a marine flavobacteria whose genome has recently been sequenced. It represents a model for the interaction between algae and bacteria. Indeed, 3 % of genes are involved in polysaccharide degradation, many of them organised in operon-like structures. Moreover, many enzymes present in the genome appear to be involved in original metabolisms, such as iodine utilization. My interest focused on the molecular aspects of three different factors involved in interaction of Z. galactanivorans with algae. The genome of Z. galactanivorans revealed two new sequences of glycosides hydrolases family 82 which are particular, due to their truncated sequences. Based on multiple sequence alignments, I designed site directed mutagenesis experiments, performed with the Alteromonas fortis enzyme, the first structural representant of iota-carrageenases. The results show that E245 (proton donor) and D247 (nucleophile) are the catalytic residues and that residues Q222, H281 and E310 also play crucial roles in the enzymatic reaction. Using a crystallographic approach, I was able to highlight the importance of a chloride ion in the formation of a water network, close to the active site. I also initiated the first biochemical and structural characterization of two members of a new GH family, located in operon-like gene organisations apparently involved in the degradation of sulphated polysaccharides. A third part of my thesis concerns the crystallographic structure determination of a first prokaryote, vanadium dependent iodoperoxydase, identified in the Z. galactanivorans genome. The structural analysis of this enzyme that may be involved in a detoxification reaction following the oxidative burst generated by algae, allows us to suggest a new evolution pattern for this type of enzymes.; La surface des algues est colonisée par des bactéries marines hétérotrophes qui entretiennent avec leurs hôtes différentes relations trophiques. Zobellia galactanivorans, une flavobactérie marine, est un modèle de l'interaction entre les bactéries et les algues. 3 % des gènes de cette bactérie sont impliquées dans la dégradation de polysaccharides. De plus on retrouve, dans son génome, des enzymes participant à des métabolismes originaux comme celui de l'iode. Je me suis intéressé à 3 aspects moléculaires de l'interaction entre Z. galactanivorans et les algues. Premièrement le génome de Z. galactanivorans possède deux nouvelles iota-carraghénases de la famille GH 82, plus courtes que celles déjà décrites. Dans le but d'identifier la machinerie catalytique de ces enzymes, je me suis basé sur l'alignement multiple de l'ensemble des séquences de la famille GH 82 pour réaliser des expériences de mutagenèse dirigées sur la iotases d'Alteromonas fortis et ainsi expliquer le mécanisme catalytique de cette famille de glycoside hydrolase. J'ai également effectué une première caractérisation biochimique et structurale de deux membres d'une nouvelle famille de glycoside hydrolases, identifié, dans le génome de Z. galactanivorans, au sein d'opérons putatifs très probablement impliqués dans la dégradation de polysaccharides sulfatés. Enfin, j'ai résolu la première structure cristallographique d'une iodoperoxydase vanadium-dépendante d'origine bactérienne. Cette enzyme pourrait être impliquée dans un mécanisme de détoxification de la bactérie, suite à un ''burst oxydant'' généré par l'algue. Cette première structure permet de revoir et de discuter l'évolution de ces protéines.