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Featured researches published by David Ropartz.


Biotechnology for Biofuels | 2015

Substrate specificity and regioselectivity of fungal AA9 lytic polysaccharide monooxygenases secreted by Podospora anserina.

Chloé Bennati-Granier; Sona Garajova; Charlotte Champion; Sacha Grisel; Mireille Haon; Simeng Zhou; Mathieu Fanuel; David Ropartz; Hélène Rogniaux; Isabelle Gimbert; Eric Record; Jean-Guy Berrin

BackgroundThe understanding of enzymatic polysaccharide degradation has progressed intensely in the past few years with the identification of a new class of fungal-secreted enzymes, the lytic polysaccharide monooxygenases (LPMOs) that enhance cellulose conversion. In the fungal kingdom, saprotrophic fungi display a high number of genes encoding LPMOs from family AA9 but the functional relevance of this redundancy is not fully understood.ResultsIn this study, we investigated a set of AA9 LPMOs identified in the secretomes of the coprophilous ascomycete Podospora anserina, a biomass degrader of recalcitrant substrates. Their activity was assayed on cellulose in synergy with the cellobiose dehydrogenase from the same organism. We showed that the total release of oxidized oligosaccharides from cellulose was higher for PaLPMO9A, PaLPMO9E, and PaLPMO9H that harbored a carbohydrate-binding module from the family CBM1. Investigation of their regioselective mode of action revealed that PaLPMO9A and PaLPMO9H oxidatively cleaved at both C1 and C4 positions while PaLPMO9E released only C1-oxidized products. Rapid cleavage of cellulose was observed using PaLPMO9H that was the most versatile in terms of substrate specificity as it also displayed activity on cello-oligosaccharides and β-(1,4)-linked hemicellulose polysaccharides (e.g., xyloglucan, glucomannan).ConclusionsThis study provides insights into the mode of cleavage and substrate specificities of fungal AA9 LPMOs that will facilitate their application for the development of future biorefineries.


The Plant Cell | 2013

PECTIN METHYLESTERASE INHIBITOR6 Promotes Arabidopsis Mucilage Release by Limiting Methylesterification of Homogalacturonan in Seed Coat Epidermal Cells

Susana Saez-Aguayo; Marie-Christine Ralet; Adeline Berger; Lucy Botran; David Ropartz; Annie Marion-Poll; Helen M. North

Djarly, a natural Arabidopsis mutant defective in seed mucilage release, is shown to be defective in a pectin methylesterase inhibitor, PMEI6. Mutant and overexpressor phenotypes highlight the importance of modulating the degree of homogalacturonan methylesterification for correct primary cell wall fragmentation and pectin partitioning into adherent and soluble mucilage layers. Imbibed seeds of the Arabidopsis thaliana accession Djarly are affected in mucilage release from seed coat epidermal cells. The impaired locus was identified as a pectin methylesterase inhibitor gene, PECTIN METHYLESTERASE INHIBITOR6 (PMEI6), specifically expressed in seed coat epidermal cells at the time when mucilage polysaccharides are accumulated. This spatio-temporal regulation appears to be modulated by GLABRA2 and LEUNIG HOMOLOG/MUCILAGE MODIFIED1, as expression of PMEI6 is reduced in mutants of these transcription regulators. In pmei6, mucilage release was delayed and outer cell walls of epidermal cells did not fragment. Pectin methylesterases (PMEs) demethylate homogalacturonan (HG), and the majority of HG found in wild-type mucilage was in fact derived from outer cell wall fragments. This correlated with the absence of methylesterified HG labeling in pmei6, whereas transgenic plants expressing the PMEI6 coding sequence under the control of the 35S promoter had increased labeling of cell wall fragments. Activity tests on seeds from pmei6 and 35S:PMEI6 transgenic plants showed that PMEI6 inhibits endogenous PME activities, in agreement with reduced overall methylesterification of mucilage fractions and demucilaged seeds. Another regulator of PME activity in seed coat epidermal cells, the subtilisin-like Ser protease SBT1.7, acts on different PMEs, as a pmei6 sbt1.7 mutant showed an additive phenotype.


Scientific Reports | 2016

Single-domain flavoenzymes trigger lytic polysaccharide monooxygenases for oxidative degradation of cellulose

Sona Garajova; Yann Mathieu; Maria Rosa Beccia; Chloé Bennati-Granier; Frédéric Biaso; Mathieu Fanuel; David Ropartz; Bruno Guigliarelli; Eric Record; Hélène Rogniaux; Bernard Henrissat; Jean-Guy Berrin

The enzymatic conversion of plant biomass has been recently revolutionized by the discovery of lytic polysaccharide monooxygenases (LPMOs) that carry out oxidative cleavage of polysaccharides. These very powerful enzymes are abundant in fungal saprotrophs. LPMOs require activation by electrons that can be provided by cellobiose dehydrogenases (CDHs), but as some fungi lack CDH-encoding genes, other recycling enzymes must exist. We investigated the ability of AA3_2 flavoenzymes secreted under lignocellulolytic conditions to trigger oxidative cellulose degradation by AA9 LPMOs. Among the flavoenzymes tested, we show that glucose dehydrogenase and aryl-alcohol quinone oxidoreductases are catalytically efficient electron donors for LPMOs. These single-domain flavoenzymes display redox potentials compatible with electron transfer between partners. Our findings extend the array of enzymes which regulate the oxidative degradation of cellulose by lignocellulolytic fungi.


Protein Engineering Design & Selection | 2014

Semi-rational approach for converting a GH1 β-glycosidase into a β-transglycosidase

David Teze; Johann Hendrickx; Mirjam Czjzek; David Ropartz; Yves-Henri Sanejouand; Vinh Tran; Charles Tellier; Michel Dion

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.


Journal of Experimental Botany | 2014

New insights into the structural and spatial variability of cell-wall polysaccharides during wheat grain development, as revealed through MALDI mass spectrometry imaging

Dušan Veličković; David Ropartz; Fabienne Guillon; Luc Saulnier; Hélène Rogniaux

Arabinoxylans (AX) and (1→3),(1→4)-β-glucans (BG) are the major components of wheat grain cell walls. Although incompletely described at the molecular level, it is known that the chemical and distributional heterogeneity of these compounds impacts the quality and use of wheat. In this work, an emerging technique based on MALDI mass spectrometry imaging (MSI) was employed to map variations in the quantity, localization, and structure of these polysaccharides in the endosperm during wheat maturation. MALDI MSI couples detailed structural information with the spatial localization observed at the micrometer scale. The enzymic hydrolysis of AX and BG was performed directly on the grain sections, resulting in the efficient formation of smaller oligosaccharides that are easily measurable through MS, with no relocation across the grain. The relative quantification of the generated oligosaccharides was achieved. The method was validated by confirming data previously obtained using other analytical techniques. Furthermore, in situ analysis of grain cell walls through MSI revealed previously undetectable intense acetylation of AX in young compared to mature grains, together with findings concerning the feruloylation of AX and different structural features of BG. These results provide new insights into the physiological roles of these polysaccharides in cell walls and the specificity of the hydrolytic enzymes involved.


Biomacromolecules | 2012

Innovative enzymatic approach to resolve homogalacturonans based on their methylesterification pattern.

Marie-Christine Ralet; Martin A. K. Williams; Abrisham Tanhatan-Nasseri; David Ropartz; Bernard Quemener; Estelle Bonnin

Three series of model homogalacturonans (HGs) covering a large range of degree of methylesterification (DM) were prepared by chemical and/or enzymatic means. Randomly demethylesterified HGs, HGs containing a few long demethylesterified galacturonic acid stretches, and HGs with numerous but short demethylesterified blocks were recovered. The analysis of the degradation products generated by the action of a purified pectin lyase allowed the definition of two new parameters, the degree of blockiness, and the absolute degree of blockiness of the highly methylesterified stretches (DBMe and DB(abs)Me, respectively). By combining this information with that obtained by the more traditional endopolygalacturonase digestion, the total proportion of degradable zones for a given DM was measured and was shown to permit a clear differentiation of the three types of HG series over a large range of DM. This double enzymatic approach will be of interest to discriminate industrial pectin samples exhibiting different functionalities and to evaluate pectin fine structure dynamics in vivo in the plant cell wall, where pectin plays a key mechanical role.


Rapid Communications in Mass Spectrometry | 2011

Performance evaluation on a wide set of matrix‐assisted laser desorption ionization matrices for the detection of oligosaccharides in a high‐throughput mass spectrometric screening of carbohydrate depolymerizing enzymes

David Ropartz; Pierre-Edouard Bodet; Cédric Przybylski; Florence Gonnet; Régis Daniel; Maude Fer; William Helbert; Dominique Bertrand; Hélène Rogniaux

Compared to other analytical methods, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) presents several unique advantages for the structural characterization of degradation products of carbohydrates. Our final goal is to implement this technique as a high-throughput platform, with the aim of exploring natural bio-diversity to discover new carbohydrate depolymerizing enzymes. In this approach, a variety of carbohydrates will be used as enzymes substrates and MALDI-MS will be employed to monitor the oligosaccharides produced. One drawback of MALDI, however, is that the choice of the matrix is largely dependent on the chemical properties of the analyte. In this context, our objective in the present work was to find the smallest set of MALDI matrices able to detect chemically heterogeneous oligosaccharides. This was done through the performance evaluation of more than 40 MALDI matrices preparations. Homogeneity of analyte-matrix deposits was considered as a critical feature, especially since the final objective is to fully automate the analyses. Evaluation of the matrices was done by means of a rigorous statistical approach. Amongst all tested compounds, our work proposes the use of the DHB/DMA ionic matrix as the most generic matrix, for rapid detection of a variety of polysaccharides including neutral, anionic, methylated, sulfated, and acetylated compounds. The selected matrices were then used to screen crude bacterial incubation media for the detection of enzymatic degradation products.


Carbohydrate Polymers | 2012

Structural data on a bacterial exopolysaccharide produced by a deep-sea Alteromonas macleodii strain

T. Le Costaouëc; S. Cérantola; David Ropartz; Jacqueline Ratiskol; Corinne Sinquin; Sylvia Colliec-Jouault; Claire Boisset

Some marine bacteria collected around deep-sea hydrothermal vents are able to produce, in laboratory conditions, complex and innovative exopolysaccharides. In a previous study, the mesophilic strain Alteromonas macleodii subsp. fijiensis biovar deepsane was collected on the East Pacific Rise at 2600 m depth. It was isolated from a polychaete annelid Alvinella pompejana and is able to synthesise and excrete the exopolysaccharide deepsane. Biological activities have been screened and some protective properties have been established. Deepsane is commercially available in cosmetics under the name of Abyssine(®) for soothing and reducing irritation of sensitive skin against chemical, mechanical and UVB aggression. This study presents structural data for this original and complex bacterial exopolysaccharide and highlights some structural similarities with other known EPS produced by marine Alteromonas strains.


Journal of Biological Chemistry | 2011

α-Galactosidase/sucrose kinase (AgaSK), a novel bifunctional enzyme from the human microbiome coupling galactosidase and kinase activities.

Laëtitia Bruel; Gerlind Sulzenbacher; Marine Cervera Tison; Ange Pujol; Cendrine Nicoletti; Josette Perrier; Anne Galinier; David Ropartz; Michel Fons; Frédérique Pompeo; Thierry Giardina

Background: Raffinose, an abundant carbohydrate in plants, is degraded into galactose and sucrose by intestinal microbial enzymes. Results: AgaSK is a protein coupling galactosidase and sucrose kinase activity. The structure of the galactosidase domain sheds light onto substrate recognition. Conclusion: AgaSK produces sucrose-6-phosphate directly from raffinose. Significance: Production of sucrose-6-phosphate directly from raffinose points toward a novel glycolytic pathway in bacteria. α-Galactosides are non-digestible carbohydrates widely distributed in plants. They are a potential source of energy in our daily food, and their assimilation by microbiota may play a role in obesity. In the intestinal tract, they are degraded by microbial glycosidases, which are often modular enzymes with catalytic domains linked to carbohydrate-binding modules. Here we introduce a bifunctional enzyme from the human intestinal bacterium Ruminococcus gnavus E1, α-galactosidase/sucrose kinase (AgaSK). Sequence analysis showed that AgaSK is composed of two domains: one closely related to α-galactosidases from glycoside hydrolase family GH36 and the other containing a nucleotide-binding motif. Its biochemical characterization showed that AgaSK is able to hydrolyze melibiose and raffinose to galactose and either glucose or sucrose, respectively, and to specifically phosphorylate sucrose on the C6 position of glucose in the presence of ATP. The production of sucrose-6-P directly from raffinose points toward a glycolytic pathway in bacteria, not described so far. The crystal structures of the galactosidase domain in the apo form and in complex with the product shed light onto the reaction and substrate recognition mechanisms and highlight an oligomeric state necessary for efficient substrate binding and suggesting a cross-talk between the galactose and kinase domains.


Nature Communications | 2017

Carrageenan catabolism is encoded by a complex regulon in marine heterotrophic bacteria

Elizabeth Ficko-Blean; Aurélie Préchoux; François Thomas; Tatiana Rochat; Robert Larocque; Yongtao Zhu; Mark Stam; Sabine Génicot; Murielle Jam; Alexandra Calteau; Benjamin Viart; David Ropartz; David Pérez-Pascual; Gaëlle Correc; Maria Matard-Mann; Keith A. Stubbs; Hélène Rogniaux; Alexandra Jeudy; Tristan Barbeyron; Claudine Médigue; Mirjam Czjzek; David Vallenet; Mark J. McBride; Eric Duchaud; Gurvan Michel

Macroalgae contribute substantially to primary production in coastal ecosystems. Their biomass, mainly consisting of polysaccharides, is cycled into the environment by marine heterotrophic bacteria using largely uncharacterized mechanisms. Here we describe the complete catabolic pathway for carrageenans, major cell wall polysaccharides of red macroalgae, in the marine heterotrophic bacterium Zobellia galactanivorans. Carrageenan catabolism relies on a multifaceted carrageenan-induced regulon, including a non-canonical polysaccharide utilization locus (PUL) and genes distal to the PUL, including a susCD-like pair. The carrageenan utilization system is well conserved in marine Bacteroidetes but modified in other phyla of marine heterotrophic bacteria. The core system is completed by additional functions that might be assumed by non-orthologous genes in different species. This complex genetic structure may be the result of multiple evolutionary events including gene duplications and horizontal gene transfers. These results allow for an extension on the definition of bacterial PUL-mediated polysaccharide digestion.Carrageenans, major cell wall polysaccharides of red macroalgae, are metabolised by marine heterotrophic bacteria through unclear mechanisms. Here, the authors identify an unusual polysaccharide-utilization locus encoding carrageenan catabolism in a marine bacterium, and characterise the complete pathway.

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Hélène Rogniaux

Institut national de la recherche agronomique

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Mathieu Fanuel

Institut national de la recherche agronomique

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Alexandre Giuliani

Institut national de la recherche agronomique

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Fabienne Guillon

Institut national de la recherche agronomique

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Marie-Christine Ralet

Institut national de la recherche agronomique

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Eric Record

Aix-Marseille University

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Sona Garajova

Aix-Marseille University

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