Charles TELLIER
Professeur émérite Université
section 64
| Équipe : |
Thèmes de recherche
Ingénierie des protéines, glycoenzymes
Projets
Parcours universitaire
Doctorat es-sciences (1986)
Publications
1 publication
Saumonneau, Amélie; Lagneau, Nathan; Ogonda, Lydia Awuor; Dupré, Catherine; Dutertre, Stéphanie; Grizeau, Dominique; Tellier, Charles; Grandjean, Cyrille; Daligault, Franck
Disruption of Botryococcus braunii colonies by glycoside hydrolases Article de journal
Dans: Bioresource Technology Reports, vol. 21, p. 101335, 2023, ISSN: 2589-014X.
@article{SAUMONNEAU2023101335,
title = {Disruption of Botryococcus braunii colonies by glycoside hydrolases},
author = {Amélie Saumonneau and Nathan Lagneau and Lydia Awuor Ogonda and Catherine Dupré and Stéphanie Dutertre and Dominique Grizeau and Charles Tellier and Cyrille Grandjean and Franck Daligault},
url = {https://www.sciencedirect.com/science/article/pii/S2589014X23000063},
doi = {10.1016/j.biteb.2023.101335},
issn = {2589-014X},
year = {2023},
date = {2023-01-13},
urldate = {2023-01-01},
journal = {Bioresource Technology Reports},
volume = {21},
pages = {101335},
abstract = {Microalgae are a promising alternative resource to fossil-based products. Botryococcus braunii is a colonial green microalga having the ability to convert CO2 by photosynthesis into long chain hydrocarbons. These are excreted and trapped in an extracellular matrix (ECM). A panel of glycosidases ranging from arabinanase, galactananase to endoglucanase was tested for their ability to lyse the polysaccharides maintaining the B. braunii colony integrity in order to release the hydrocarbons present in the extracellular matrix without harming the cells. The BpGH9 endoglucanase from Bacillus pumilus was fused with CtCBM3a from Clostridium thermocellum and yellow fluorescent protein to probe the presence of microcrystalline cellulose in the cell wall of B. braunii and to increase the efficacy of the endoglucanase. All the tested enzymes were able to some extent to dissociate the cells from the extracellular matrix while keeping them alive, suggesting the feasibility of a semi-continuous in situ recovery of hydrocarbons.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Microalgae are a promising alternative resource to fossil-based products. Botryococcus braunii is a colonial green microalga having the ability to convert CO2 by photosynthesis into long chain hydrocarbons. These are excreted and trapped in an extracellular matrix (ECM). A panel of glycosidases ranging from arabinanase, galactananase to endoglucanase was tested for their ability to lyse the polysaccharides maintaining the B. braunii colony integrity in order to release the hydrocarbons present in the extracellular matrix without harming the cells. The BpGH9 endoglucanase from Bacillus pumilus was fused with CtCBM3a from Clostridium thermocellum and yellow fluorescent protein to probe the presence of microcrystalline cellulose in the cell wall of B. braunii and to increase the efficacy of the endoglucanase. All the tested enzymes were able to some extent to dissociate the cells from the extracellular matrix while keeping them alive, suggesting the feasibility of a semi-continuous in situ recovery of hydrocarbons.
2 publications
Assailly, Coralie; Bridot, Clarisse; Saumonneau, Amélie; Lottin, Paul; Roubinet, Benoit; Krammer, Eva-Maria; François, Francesca; Vena, Federica; Landemarre, Ludovic; Dorta, Dimitri Alvarez; Deniaud, David; Grandjean, Cyrille; Tellier, Charles; Pascual, Sagrario; Montembault, Véronique; Fontaine, Laurent; Daligault, Franck; Bouckaert, Julie; Gouin, Sébastien G
Polyvalent Transition-State Analogues of Sialyl Substrates Strongly Inhibit Bacterial Sialidases** Article de journal
Dans: Chemistry – A European Journal, vol. 27, no. 9, p. 3142-3150, 2021.
@article{https://doi.org/10.1002/chem.202004672,
title = {Polyvalent Transition-State Analogues of Sialyl Substrates Strongly Inhibit Bacterial Sialidases**},
author = {Coralie Assailly and Clarisse Bridot and Amélie Saumonneau and Paul Lottin and Benoit Roubinet and Eva-Maria Krammer and Francesca François and Federica Vena and Ludovic Landemarre and Dimitri Alvarez Dorta and David Deniaud and Cyrille Grandjean and Charles Tellier and Sagrario Pascual and Véronique Montembault and Laurent Fontaine and Franck Daligault and Julie Bouckaert and Sébastien G Gouin},
url = {https://chemistry-europe.onlinelibrary.wiley.com/doi/abs/10.1002/chem.202004672},
doi = {https://doi.org/10.1002/chem.202004672},
year = {2021},
date = {2021-01-01},
journal = {Chemistry – A European Journal},
volume = {27},
number = {9},
pages = {3142-3150},
abstract = {Abstract Bacterial sialidases (SA) are validated drug targets expressed by common human pathogens such as Streptococcus pneumoniae, Vibrio cholerae, or Clostridium perfringens. Noncovalent inhibitors of bacterial SA capable of reaching the submicromolar level are rarely reported. In this work, multi- and polyvalent compounds are developed, based on the transition-state analogue 2-deoxy-2,3-didehydro-N-acetylneuraminic (DANA). Poly-DANA inhibits the catalytic activity of SA from S. pneumoniae (NanA) and the symbiotic microorganism B. thetaiotaomicron (BtSA) at the picomolar and low nanomolar levels (expressed in moles of molecules and of DANA, respectively). Each DANA grafted to the polymer surpasses the inhibitory potential of the monovalent analogue by more than four orders of magnitude, which represents the highest multivalent effect reported so far for an enzyme inhibition. The synergistic interaction is shown to operate exclusively in the catalytic domain, and not in the flanked carbohydrate-binding module (CBM). These results offer interesting perspectives for the multivalent inhibition of other SA families lacking a CBM, such as viral, parasitic, or human SA.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Abstract Bacterial sialidases (SA) are validated drug targets expressed by common human pathogens such as Streptococcus pneumoniae, Vibrio cholerae, or Clostridium perfringens. Noncovalent inhibitors of bacterial SA capable of reaching the submicromolar level are rarely reported. In this work, multi- and polyvalent compounds are developed, based on the transition-state analogue 2-deoxy-2,3-didehydro-N-acetylneuraminic (DANA). Poly-DANA inhibits the catalytic activity of SA from S. pneumoniae (NanA) and the symbiotic microorganism B. thetaiotaomicron (BtSA) at the picomolar and low nanomolar levels (expressed in moles of molecules and of DANA, respectively). Each DANA grafted to the polymer surpasses the inhibitory potential of the monovalent analogue by more than four orders of magnitude, which represents the highest multivalent effect reported so far for an enzyme inhibition. The synergistic interaction is shown to operate exclusively in the catalytic domain, and not in the flanked carbohydrate-binding module (CBM). These results offer interesting perspectives for the multivalent inhibition of other SA families lacking a CBM, such as viral, parasitic, or human SA.
Ogonda, Lydia A; Saumonneau, Amélie; Dion, Michel; Muge, Edward K; Wamalwa, Benson M; Mulaa, Francis J; Tellier, Charles
Characterization and engineering of two new GH9 and GH48 cellulases from a Bacillus pumilus isolated from Lake Bogoria Article de journal
Dans: Biotechnology Letters, vol. 43, p. 691–700, 2021.
@article{ogondacharacterization,
title = {Characterization and engineering of two new GH9 and GH48 cellulases from a Bacillus pumilus isolated from Lake Bogoria},
author = {Lydia A Ogonda and Amélie Saumonneau and Michel Dion and Edward K Muge and Benson M Wamalwa and Francis J Mulaa and Charles Tellier},
doi = {10.1007/s10529-020-03056-z},
year = {2021},
date = {2021-01-01},
journal = {Biotechnology Letters},
volume = {43},
pages = {691–700},
publisher = {Springer},
abstract = {Objectives. To search for new alkaliphilic cellulases and to improve their efficiency on crystalline cellulose through molecular engineering
Results. Two novel cellulases, BpGH9 and BpGH48, from a Bacillus pumilus strain were identified, cloned and biochemically characterized. BpGH9 is a modular endocellulase belonging to the glycoside hydrolase 9 family (GH9), which contains a catalytic module (GH) and a carbohydrate-binding module belonging to class 3 and subclass c (CBM3c). This enzyme is extremely tolerant to high alkali pH and remains significantly active at pH 10. BpGH48 is an exocellulase, belonging to the glycoside hydrolase 48 family (GH48) and acts on the reducing end of oligo-β1,4 glucanes. A truncated form of BpGH9 and a chimeric fusion with an additional CBM3a module was constructed. The deletion of the CBM3c module results in a significant decline in the catalytic activity. However, fusion of CBM3a, although in a non native position, enhanced the activity of BpGH9 on crystalline cellulose.
Conclusions. A new alkaliphilic endocellulase BpGH9, was cloned and engineered as a fusion protein (CBM3a-BpGH9), which led to an improved activity on crystalline cellulose.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Objectives. To search for new alkaliphilic cellulases and to improve their efficiency on crystalline cellulose through molecular engineering
Results. Two novel cellulases, BpGH9 and BpGH48, from a Bacillus pumilus strain were identified, cloned and biochemically characterized. BpGH9 is a modular endocellulase belonging to the glycoside hydrolase 9 family (GH9), which contains a catalytic module (GH) and a carbohydrate-binding module belonging to class 3 and subclass c (CBM3c). This enzyme is extremely tolerant to high alkali pH and remains significantly active at pH 10. BpGH48 is an exocellulase, belonging to the glycoside hydrolase 48 family (GH48) and acts on the reducing end of oligo-β1,4 glucanes. A truncated form of BpGH9 and a chimeric fusion with an additional CBM3a module was constructed. The deletion of the CBM3c module results in a significant decline in the catalytic activity. However, fusion of CBM3a, although in a non native position, enhanced the activity of BpGH9 on crystalline cellulose.
Conclusions. A new alkaliphilic endocellulase BpGH9, was cloned and engineered as a fusion protein (CBM3a-BpGH9), which led to an improved activity on crystalline cellulose.
Results. Two novel cellulases, BpGH9 and BpGH48, from a Bacillus pumilus strain were identified, cloned and biochemically characterized. BpGH9 is a modular endocellulase belonging to the glycoside hydrolase 9 family (GH9), which contains a catalytic module (GH) and a carbohydrate-binding module belonging to class 3 and subclass c (CBM3c). This enzyme is extremely tolerant to high alkali pH and remains significantly active at pH 10. BpGH48 is an exocellulase, belonging to the glycoside hydrolase 48 family (GH48) and acts on the reducing end of oligo-β1,4 glucanes. A truncated form of BpGH9 and a chimeric fusion with an additional CBM3a module was constructed. The deletion of the CBM3c module results in a significant decline in the catalytic activity. However, fusion of CBM3a, although in a non native position, enhanced the activity of BpGH9 on crystalline cellulose.
Conclusions. A new alkaliphilic endocellulase BpGH9, was cloned and engineered as a fusion protein (CBM3a-BpGH9), which led to an improved activity on crystalline cellulose.
2 publications
Teze, David; Coines, Joan; Raich, Lluís; Kalichuk, Valentina; Solleux, Claude; Tellier, Charles; André-Miral, Corinne; Svensson, Birte; Rovira, Carme
A Single Point Mutation Converts GH84 O-GlcNAc Hydrolases into Phosphorylases: Experimental and Theoretical Evidence Article de journal
Dans: Journal of the American Chemical Society, vol. 142, no. 5, p. 2120–2124, 2020, ISSN: 15205126.
@article{Teze2020,
title = {A Single Point Mutation Converts GH84 O-GlcNAc Hydrolases into Phosphorylases: Experimental and Theoretical Evidence},
author = {David Teze and Joan Coines and Lluís Raich and Valentina Kalichuk and Claude Solleux and Charles Tellier and Corinne André-Miral and Birte Svensson and Carme Rovira},
doi = {10.1021/jacs.9b09655},
issn = {15205126},
year = {2020},
date = {2020-01-01},
journal = {Journal of the American Chemical Society},
volume = {142},
number = {5},
pages = {2120--2124},
abstract = {Glycoside hydrolases and phosphorylases are two major classes of enzymes responsible for the cleavage of glycosidic bonds. Here we show that two GH84 O-GlcNAcase enzymes can be converted to efficient phosphorylases by a single point mutation. Noteworthy, the mutated enzymes are over 10-fold more active than naturally occurring glucosaminide phosphorylases. We rationalize this novel transformation using molecular dynamics and QM/MM metadynamics methods, showing that the mutation changes the electrostatic potential at the active site and reduces the energy barrier for phosphorolysis by 10 kcaltextperiodcenteredmol-1. In addition, the simulations unambiguously reveal the nature of the intermediate as a glucose oxazolinium ion, clarifying the debate on the nature of such a reaction intermediate in glycoside hydrolases operating via substrate-assisted catalysis.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Glycoside hydrolases and phosphorylases are two major classes of enzymes responsible for the cleavage of glycosidic bonds. Here we show that two GH84 O-GlcNAcase enzymes can be converted to efficient phosphorylases by a single point mutation. Noteworthy, the mutated enzymes are over 10-fold more active than naturally occurring glucosaminide phosphorylases. We rationalize this novel transformation using molecular dynamics and QM/MM metadynamics methods, showing that the mutation changes the electrostatic potential at the active site and reduces the energy barrier for phosphorolysis by 10 kcaltextperiodcenteredmol-1. In addition, the simulations unambiguously reveal the nature of the intermediate as a glucose oxazolinium ion, clarifying the debate on the nature of such a reaction intermediate in glycoside hydrolases operating via substrate-assisted catalysis.
Violo, Typhaine; Dussouy, Christophe; Tellier, Charles; Grandjean, Cyrille; Camberlein, Emilie
Homogenous Glycoconjugate Produced by Combined Unnatural Amino Acid Incorporation and Click-Chemistry for Vaccine Purposes Article de journal
Dans: Journal of visualized experiments : JoVE, 2020.
@article{violo:hal-02990572,
title = {Homogenous Glycoconjugate Produced by Combined Unnatural Amino Acid Incorporation and Click-Chemistry for Vaccine Purposes},
author = {Typhaine Violo and Christophe Dussouy and Charles Tellier and Cyrille Grandjean and Emilie Camberlein},
url = {https://hal.archives-ouvertes.fr/hal-02990572},
doi = {10.3791/60821},
year = {2020},
date = {2020-01-01},
journal = {Journal of visualized experiments : JoVE},
publisher = {JoVE},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
3 publications
Brissonnet, Yoan; Assailly, Coralie; Saumonneau, Amélie; Bouckaert, Julie; Maillasson, Mike; Petitot, Clémence; Roubinet, Benoit; Didak, Blanka; Landemarre, Ludovic; Bridot, Clarisse; Blossey, Ralf; Deniaud, David; Yan, Xibo; Bernard, Julien; Tellier, Charles; Grandjean, Cyrille; Daligault, Franck; Gouin, Sébastien G
Multivalent Thiosialosides and Their Synergistic Interaction with Pathogenic Sialidases Article de journal
Dans: Chemistry - A European Journal, vol. 25, no. 9, p. 2358–2365, 2019, ISSN: 15213765.
@article{Brissonnet2019b,
title = {Multivalent Thiosialosides and Their Synergistic Interaction with Pathogenic Sialidases},
author = {Yoan Brissonnet and Coralie Assailly and Amélie Saumonneau and Julie Bouckaert and Mike Maillasson and Clémence Petitot and Benoit Roubinet and Blanka Didak and Ludovic Landemarre and Clarisse Bridot and Ralf Blossey and David Deniaud and Xibo Yan and Julien Bernard and Charles Tellier and Cyrille Grandjean and Franck Daligault and Sébastien G Gouin},
doi = {10.1002/chem.201805790},
issn = {15213765},
year = {2019},
date = {2019-01-01},
journal = {Chemistry - A European Journal},
volume = {25},
number = {9},
pages = {2358--2365},
abstract = {Sialidases (SAs) hydrolyze sialyl residues from glycoconjugates of the eukaryotic cell surface and are virulence factors expressed by pathogenic bacteria, viruses, and parasites. The catalytic domains of SAs are often flanked with carbohydrate-binding module(s) previously shown to bind sialosides and to enhance enzymatic catalytic efficiency. Herein, non-hydrolyzable multivalent thiosialosides were designed as probes and inhibitors of V. cholerae, T. cruzi, and S. pneumoniae (NanA) sialidases. NanA was truncated from the catalytic and lectinic domains (NanA-L and NanA-C) to probe their respective roles upon interacting with sialylated surfaces and the synthetically designed di- and polymeric thiosialosides. The NanA-L domain was shown to fully drive NanA binding, improving affinity for the thiosialylated surface and compounds by more than two orders of magnitude. Importantly, each thiosialoside grafted onto the polymer was also shown to reduce NanA and NanA-C catalytic activity with efficiency that was 3000-fold higher than that of the monovalent thiosialoside reference. These results extend the concept of multivalency for designing potent bacterial and parasitic sialidase inhibitors.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Sialidases (SAs) hydrolyze sialyl residues from glycoconjugates of the eukaryotic cell surface and are virulence factors expressed by pathogenic bacteria, viruses, and parasites. The catalytic domains of SAs are often flanked with carbohydrate-binding module(s) previously shown to bind sialosides and to enhance enzymatic catalytic efficiency. Herein, non-hydrolyzable multivalent thiosialosides were designed as probes and inhibitors of V. cholerae, T. cruzi, and S. pneumoniae (NanA) sialidases. NanA was truncated from the catalytic and lectinic domains (NanA-L and NanA-C) to probe their respective roles upon interacting with sialylated surfaces and the synthetically designed di- and polymeric thiosialosides. The NanA-L domain was shown to fully drive NanA binding, improving affinity for the thiosialylated surface and compounds by more than two orders of magnitude. Importantly, each thiosialoside grafted onto the polymer was also shown to reduce NanA and NanA-C catalytic activity with efficiency that was 3000-fold higher than that of the monovalent thiosialoside reference. These results extend the concept of multivalency for designing potent bacterial and parasitic sialidase inhibitors.
Naretto, Anaïs; Fanuel, Mathieu; Ropartz, David; Rogniaux, Hélène; Larocque, Robert; Czjzek, Mirjam; Tellier, Charles; Michel, Gurvan
The agar-specific hydrolase ZgAgaC from the marine bacterium Zobellia galactanivorans defines a new GH16 protein subfamily Article de journal
Dans: Journal of Biological Chemistry, vol. 294, no. 17, p. 6923–6939, 2019, ISSN: 1083351X.
@article{Naretto2019,
title = {The agar-specific hydrolase ZgAgaC from the marine bacterium Zobellia galactanivorans defines a new GH16 protein subfamily},
author = {Anaïs Naretto and Mathieu Fanuel and David Ropartz and Hélène Rogniaux and Robert Larocque and Mirjam Czjzek and Charles Tellier and Gurvan Michel},
doi = {10.1074/jbc.RA118.006609},
issn = {1083351X},
year = {2019},
date = {2019-01-01},
journal = {Journal of Biological Chemistry},
volume = {294},
number = {17},
pages = {6923--6939},
abstract = {Agars are sulfated galactans from red macroalgae and are composed of a D-galactose (G unit) and L-galactose (L unit) alternatively linked by α-1, 3 and β-1, 4 glycosidicbonds. The sepolysaccharides display high complexity, with numerous modifications of their backbone (e.g. presence of a 3, 6-anhydro-bridge (LA unit) and sulfations and methylation). Currently, bacterial polysaccharidases that hydrolyze agars (α-agarases and α-porphyranases) have been characterized on simple agarose and more rarely on porphyran, a polymer containing both agarobiose (G-LA) and porphyranobiose (GL6S) motifs. How bacteria can degrade complex agars remains therefore an open question. Here, we studied an enzyme from the marine bacterium Zobellia galactanivorans (ZgAgaC) that is distantly related to the glycoside hydrolase 16 (GH16) family α-agarases and α-porphyranases. Using a large red algae collection, we demonstrate that ZgAgaC hydrolyzes not only agarose but also complex agars from Ceramiales species. Using tandem MS analysis, we elucidated the structure of a purified hexasaccharide product, L6S-G-LA2Me-G(2Pentose)-LA2S-G, released by the activity of ZgAgaC on agar extracted from Osmundea pinnatifida. By resolving the crystal structure of ZgAgaC at high resolution (1.3 Å) and comparison with the structures of ZgAgaB and ZgPorA in complex with their respective substrates, we determined that ZgAgaC recognizes agarose via a mechanism different from that of classical α-agarases. Moreover, we identified conserved residues involved in the binding of complex oligoagars and demonstrate a probable influence of the acidic polysaccharide's pH microenvironment on hydrolase activity. Finally, a phylogenetic analysis supported the notion that ZgAgaC homologs define a new GH16 subfamily distinct from α-porphyranases and classical α-agarases.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Agars are sulfated galactans from red macroalgae and are composed of a D-galactose (G unit) and L-galactose (L unit) alternatively linked by α-1, 3 and β-1, 4 glycosidicbonds. The sepolysaccharides display high complexity, with numerous modifications of their backbone (e.g. presence of a 3, 6-anhydro-bridge (LA unit) and sulfations and methylation). Currently, bacterial polysaccharidases that hydrolyze agars (α-agarases and α-porphyranases) have been characterized on simple agarose and more rarely on porphyran, a polymer containing both agarobiose (G-LA) and porphyranobiose (GL6S) motifs. How bacteria can degrade complex agars remains therefore an open question. Here, we studied an enzyme from the marine bacterium Zobellia galactanivorans (ZgAgaC) that is distantly related to the glycoside hydrolase 16 (GH16) family α-agarases and α-porphyranases. Using a large red algae collection, we demonstrate that ZgAgaC hydrolyzes not only agarose but also complex agars from Ceramiales species. Using tandem MS analysis, we elucidated the structure of a purified hexasaccharide product, L6S-G-LA2Me-G(2Pentose)-LA2S-G, released by the activity of ZgAgaC on agar extracted from Osmundea pinnatifida. By resolving the crystal structure of ZgAgaC at high resolution (1.3 Å) and comparison with the structures of ZgAgaB and ZgPorA in complex with their respective substrates, we determined that ZgAgaC recognizes agarose via a mechanism different from that of classical α-agarases. Moreover, we identified conserved residues involved in the binding of complex oligoagars and demonstrate a probable influence of the acidic polysaccharide's pH microenvironment on hydrolase activity. Finally, a phylogenetic analysis supported the notion that ZgAgaC homologs define a new GH16 subfamily distinct from α-porphyranases and classical α-agarases.
David, Benoit; Arnaud, Philippe; Tellier, Charles; Sanejouand, Yves-Henri
Toward the design of efficient transglycosidases: the case of the GH1 of Thermus thermophilus Article de journal
Dans: Protein Engineering, Design and Selection, vol. 32, no. 7, p. 309–316, 2019, ISSN: 1741-0126.
@article{10.1093/protein/gzz032,
title = {Toward the design of efficient transglycosidases: the case of the GH1 of Thermus thermophilus},
author = {Benoit David and Philippe Arnaud and Charles Tellier and Yves-Henri Sanejouand},
url = {https://doi.org/10.1093/protein/gzz032},
doi = {10.1093/protein/gzz032},
issn = {1741-0126},
year = {2019},
date = {2019-01-01},
journal = {Protein Engineering, Design and Selection},
volume = {32},
number = {7},
pages = {309--316},
abstract = {Using the information available in the sequences of well-characterized transglycosidases found in plants, mutations were introduced in the glycoside hydrolase of the bacterium Thermus thermophilus, with the aim of turning it into an efficient transglycosidase. All mutants happen to have fair catalytic efficiencies, being at worst 25 times less efficient than the wild type. Noteworthy, W120F, one of our high transglycosylation yield (≈ 50%) mutants, is only two times less efficient than the wild type. Interestingly, while in the wild type the sidechain of the acid–base is only found able to sample a pair of equivalent conformations during 0.5-µs-long molecular dynamics simulations, its flexibility is much higher in the case of the high transglycosylation yield mutants. Our results thus suggest that engineering the flexibility of the acid–base of a retaining glycoside hydrolase could be a general way to turn it into an efficient transglycosidase.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Using the information available in the sequences of well-characterized transglycosidases found in plants, mutations were introduced in the glycoside hydrolase of the bacterium Thermus thermophilus, with the aim of turning it into an efficient transglycosidase. All mutants happen to have fair catalytic efficiencies, being at worst 25 times less efficient than the wild type. Noteworthy, W120F, one of our high transglycosylation yield (≈ 50%) mutants, is only two times less efficient than the wild type. Interestingly, while in the wild type the sidechain of the acid–base is only found able to sample a pair of equivalent conformations during 0.5-µs-long molecular dynamics simulations, its flexibility is much higher in the case of the high transglycosylation yield mutants. Our results thus suggest that engineering the flexibility of the acid–base of a retaining glycoside hydrolase could be a general way to turn it into an efficient transglycosidase.
5 publications
Legentil, Laurent; Cabezas, Yari; Tasseau, Olivier; Tellier, Charles; Daligault, Franck; Ferrières, Vincent
Regioselective Galactofuranosylation for the Synthesis of Disaccharide Patterns Found in Pathogenic Microorganisms Article de journal
Dans: Journal of Organic Chemistry, vol. 82, no. 14, p. 7114–7122, 2017, ISSN: 15206904.
@article{Legentil2017,
title = {Regioselective Galactofuranosylation for the Synthesis of Disaccharide Patterns Found in Pathogenic Microorganisms},
author = {Laurent Legentil and Yari Cabezas and Olivier Tasseau and Charles Tellier and Franck Daligault and Vincent Ferri{è}res},
doi = {10.1021/acs.joc.7b00565},
issn = {15206904},
year = {2017},
date = {2017-01-01},
journal = {Journal of Organic Chemistry},
volume = {82},
number = {14},
pages = {7114--7122},
abstract = {Koenigs-Knorr glycosylation of acceptors with more than one free hydroxyl group by 2,3,5,6-tetrabenzoyl galactofuranosyl bromide was performed using diphenylborinic acid 2-aminoethyl ester (DPBA) as inducer of regioselectivity. High regioselectivity for the glycosylation on the equatorial hydroxyl group of the acceptor was obtained thanks to the transient formation of a borinate adduct of the corresponding 1,2-cis diol. Nevertheless formation of orthoester byproducts hampered the efficiency of the method. Interestingly electron-withdrawing groups on O-6 or on C-1 of the acceptor displaced the reaction in favor of the desired galactofuranosyl containing disaccharide. The best yield was obtained for the furanosylation of p-nitrophenyl 6-O-acetyl mannopyranoside. Precursors of other disaccharides, found in the glycocalix of some pathogens, were synthesized according to the same protocol with yields ranging from 45 to 86%. This is a good alternative for the synthesis of biologically relevant glycoconjugates.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Koenigs-Knorr glycosylation of acceptors with more than one free hydroxyl group by 2,3,5,6-tetrabenzoyl galactofuranosyl bromide was performed using diphenylborinic acid 2-aminoethyl ester (DPBA) as inducer of regioselectivity. High regioselectivity for the glycosylation on the equatorial hydroxyl group of the acceptor was obtained thanks to the transient formation of a borinate adduct of the corresponding 1,2-cis diol. Nevertheless formation of orthoester byproducts hampered the efficiency of the method. Interestingly electron-withdrawing groups on O-6 or on C-1 of the acceptor displaced the reaction in favor of the desired galactofuranosyl containing disaccharide. The best yield was obtained for the furanosylation of p-nitrophenyl 6-O-acetyl mannopyranoside. Precursors of other disaccharides, found in the glycocalix of some pathogens, were synthesized according to the same protocol with yields ranging from 45 to 86%. This is a good alternative for the synthesis of biologically relevant glycoconjugates.
Dion, Johann; Deshayes, Frédérique; Storozhylova, Nataliya; Advedissian, Tamara; Lambert, Annie; Viguier, Mireille; Tellier, Charles; Dussouy, Christophe; Poirier, Françoise; Grandjean, Cyrille
Lactosamine-Based Derivatives as Tools to Delineate the Biological Functions of Galectins: Application to Skin Tissue Repair Article de journal
Dans: ChemBioChem, vol. 18, no. 8, p. 782–789, 2017, ISSN: 14397633.
@article{Dion2017c,
title = {Lactosamine-Based Derivatives as Tools to Delineate the Biological Functions of Galectins: Application to Skin Tissue Repair},
author = {Johann Dion and Frédérique Deshayes and Nataliya Storozhylova and Tamara Advedissian and Annie Lambert and Mireille Viguier and Charles Tellier and Christophe Dussouy and Françoise Poirier and Cyrille Grandjean},
doi = {10.1002/cbic.201600673},
issn = {14397633},
year = {2017},
date = {2017-01-01},
journal = {ChemBioChem},
volume = {18},
number = {8},
pages = {782--789},
abstract = {Galectins have been recognized as potential novel therapeutic targets for the numerous fundamental biological processes in which they are involved. Galectins are key players in homeostasis, and as such their expression and function are finely tuned in vivo. Thus, their modes of action are complex and remain largely unexplored, partly because of the lack of dedicated tools. We thus designed galectin inhibitors from a lactosamine core, functionalized at key C2 and C3′ positions by aromatic substituents to ensure both high affinity and selectivity, and equipped with a spacer that can be modified on demand to further modulate their physico-chemical properties. As a proof-of-concept, galectin-3 was selectively targeted. The efficacy of the synthesized di-aromatic lactosamine tools was shown in cellular assays to modulate collective epithelial cell migration and to interfere with actin/cortactin localization.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Galectins have been recognized as potential novel therapeutic targets for the numerous fundamental biological processes in which they are involved. Galectins are key players in homeostasis, and as such their expression and function are finely tuned in vivo. Thus, their modes of action are complex and remain largely unexplored, partly because of the lack of dedicated tools. We thus designed galectin inhibitors from a lactosamine core, functionalized at key C2 and C3′ positions by aromatic substituents to ensure both high affinity and selectivity, and equipped with a spacer that can be modified on demand to further modulate their physico-chemical properties. As a proof-of-concept, galectin-3 was selectively targeted. The efficacy of the synthesized di-aromatic lactosamine tools was shown in cellular assays to modulate collective epithelial cell migration and to interfere with actin/cortactin localization.
David, Benoit; Irague, Romain; Jouanneau, Diane; Daligault, Franck; Czjzek, Mirjam; Sanejouand, Yves-Henri; Tellier, Charles
Internal Water Dynamics Control the Transglycosylation/Hydrolysis Balance in the Agarase (AgaD) of Zobellia galactanivorans Article de journal
Dans: ACS Catalysis, vol. 7, no. 5, p. 3357–3367, 2017, ISSN: 21555435.
@article{David2017a,
title = {Internal Water Dynamics Control the Transglycosylation/Hydrolysis Balance in the Agarase (AgaD) of Zobellia galactanivorans},
author = {Benoit David and Romain Irague and Diane Jouanneau and Franck Daligault and Mirjam Czjzek and Yves-Henri Sanejouand and Charles Tellier},
doi = {10.1021/acscatal.7b00348},
issn = {21555435},
year = {2017},
date = {2017-01-01},
journal = {ACS Catalysis},
volume = {7},
number = {5},
pages = {3357--3367},
abstract = {In retaining glycoside hydrolases (GHs), transglycosylase activity is often low due to the natural hydrolytic activity that is favored in water. Improving the relative transglycosylase activity of these enzymes is of particular interest to obtain enzymes suitable for the synthesis of oligosaccharides. We explored the effect of engineering the water dynamics within the endo-β-agarase AgaD on the transglycosylation/hydrolysis (T/H) balance. By mutating three amino acids (D341, Q342, and S351), which could control water access to a putative water channel ending close to the active site, we obtained AgaD variants with an inverted T/H balance. For the best mutant, D341L/Q342H/S351F, the hydrolysis activity was reduced 50-fold in comparison to the wild type, while the transglycosylase activity was maintained and even slightly improved. This variant produced a large amount of oligo-agaroses by a disproportionation reaction with deca-agarose as the substrate. Molecular dynamics simulations showed that these enzymatic modifications were correlated with higher water dynamics, as revealed by a marked reduction in the water survival time and a decrease in the purge time of water in a channel ending close to the active site. These results suggest that modifying the water dynamics in GHs could be a rational basis for engineering of transglycosylase activity.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In retaining glycoside hydrolases (GHs), transglycosylase activity is often low due to the natural hydrolytic activity that is favored in water. Improving the relative transglycosylase activity of these enzymes is of particular interest to obtain enzymes suitable for the synthesis of oligosaccharides. We explored the effect of engineering the water dynamics within the endo-β-agarase AgaD on the transglycosylation/hydrolysis (T/H) balance. By mutating three amino acids (D341, Q342, and S351), which could control water access to a putative water channel ending close to the active site, we obtained AgaD variants with an inverted T/H balance. For the best mutant, D341L/Q342H/S351F, the hydrolysis activity was reduced 50-fold in comparison to the wild type, while the transglycosylase activity was maintained and even slightly improved. This variant produced a large amount of oligo-agaroses by a disproportionation reaction with deca-agarose as the substrate. Molecular dynamics simulations showed that these enzymatic modifications were correlated with higher water dynamics, as revealed by a marked reduction in the water survival time and a decrease in the purge time of water in a channel ending close to the active site. These results suggest that modifying the water dynamics in GHs could be a rational basis for engineering of transglycosylase activity.
Dion, Johann; Advedissian, Tamara; Storozhylova, Nataliya; Dahbi, Samir; Lambert, Annie; Deshayes, Frédérique; Viguier, Mireille; Tellier, Charles; Poirier, Françoise; Téletchéa, Stéphane; Dussouy, Christophe; Tateno, Hiroaki; Hirabayashi, Jun; Grandjean, Cyrille
Development of a Sensitive Microarray Platform for the Ranking of Galectin Inhibitors: Identification of a Selective Galectin-3 Inhibitor Article de journal
Dans: ChemBioChem, vol. 18, no. 24, p. 2428–2440, 2017, ISSN: 14397633.
@article{Dion2017a,
title = {Development of a Sensitive Microarray Platform for the Ranking of Galectin Inhibitors: Identification of a Selective Galectin-3 Inhibitor},
author = {Johann Dion and Tamara Advedissian and Nataliya Storozhylova and Samir Dahbi and Annie Lambert and Frédérique Deshayes and Mireille Viguier and Charles Tellier and Françoise Poirier and Stéphane Téletchéa and Christophe Dussouy and Hiroaki Tateno and Jun Hirabayashi and Cyrille Grandjean},
doi = {10.1002/cbic.201700544},
issn = {14397633},
year = {2017},
date = {2017-01-01},
journal = {ChemBioChem},
volume = {18},
number = {24},
pages = {2428--2440},
abstract = {Glycan microarrays are useful tools for lectin glycan profiling. The use of a glycan microarray based on evanescent-field fluorescence detection was herein further extended to the screening of lectin inhibitors in competitive experiments. The efficacy of this approach was tested with 2/3′-mono- and 2,3′-diaromatic type II lactosamine derivatives and galectins as targets and was validated by comparison with fluorescence anisotropy proposed as an orthogonal protein interaction measurement technique. We showed that subtle differences in the architecture of the inhibitor could be sensed that pointed out the preference of galectin-3 for 2′-arylamido derivatives over ureas, thioureas, and amines and that of galectin-7 for derivatives bearing an α substituent at the anomeric position of glucosamine. We eventually identified a diaromatic oxazoline as a highly specific inhibitor of galectin-3 versus galectin-1 and galectin-7.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Glycan microarrays are useful tools for lectin glycan profiling. The use of a glycan microarray based on evanescent-field fluorescence detection was herein further extended to the screening of lectin inhibitors in competitive experiments. The efficacy of this approach was tested with 2/3′-mono- and 2,3′-diaromatic type II lactosamine derivatives and galectins as targets and was validated by comparison with fluorescence anisotropy proposed as an orthogonal protein interaction measurement technique. We showed that subtle differences in the architecture of the inhibitor could be sensed that pointed out the preference of galectin-3 for 2′-arylamido derivatives over ureas, thioureas, and amines and that of galectin-7 for derivatives bearing an α substituent at the anomeric position of glucosamine. We eventually identified a diaromatic oxazoline as a highly specific inhibitor of galectin-3 versus galectin-1 and galectin-7.
Alvarez-Dorta, Dimitri; Brissonnet, Yoan; Saumonneau, Amélie; Deniaud, David; Bernard, Julien; Yan, Xibo; Tellier, Charles; Daligault, Franck; Gouin, Sébastien G
Magnetic Nanoparticles Coated with Thiomannosides or Iminosugars to Switch and Recycle Galactosidase Activity Article de journal
Dans: ChemistrySelect, vol. 2, no. 29, p. 9552–9556, 2017, ISSN: 23656549.
@article{Alvarez-Dorta2017,
title = {Magnetic Nanoparticles Coated with Thiomannosides or Iminosugars to Switch and Recycle Galactosidase Activity},
author = {Dimitri Alvarez-Dorta and Yoan Brissonnet and Amélie Saumonneau and David Deniaud and Julien Bernard and Xibo Yan and Charles Tellier and Franck Daligault and Sébastien G Gouin},
doi = {10.1002/slct.201702063},
issn = {23656549},
year = {2017},
date = {2017-01-01},
journal = {ChemistrySelect},
volume = {2},
number = {29},
pages = {9552--9556},
abstract = {Glycosidase effectors have rarely been reported despite their great potential interest in pharmaceutical sciences and industry. Magnetic nanoparticles were coated with thiomannosides (SMan@Fe3O4) or the broad spectrum glycosidase inhibitor deoxynojirimycin (DNJ@Fe3O4). The coated ligands were shown to exert a fully reverse effect on a model galactosidase (AgaB), with SMan@Fe3O4 or DNJ@Fe3O4 ligands acting as an enzyme inhibitor (Ki=3.7 µM) or a strong activator (250% higher AgaB velocity at 50 µM), respectively. This is striking considering that monovalent soluble SMan and DNJ analogues do not interact with AgaB at millimolar concentrations. The AgaB-DNJ@Fe3O4 enzyme-effector complex could be magnetically recycled and still showed a higher activity compared to free AgaB after four catalytic cycles. The “boost and recycle” procedure may provide interesting perspectives in glycosidase biocatalysis.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Glycosidase effectors have rarely been reported despite their great potential interest in pharmaceutical sciences and industry. Magnetic nanoparticles were coated with thiomannosides (SMan@Fe3O4) or the broad spectrum glycosidase inhibitor deoxynojirimycin (DNJ@Fe3O4). The coated ligands were shown to exert a fully reverse effect on a model galactosidase (AgaB), with SMan@Fe3O4 or DNJ@Fe3O4 ligands acting as an enzyme inhibitor (Ki=3.7 µM) or a strong activator (250% higher AgaB velocity at 50 µM), respectively. This is striking considering that monovalent soluble SMan and DNJ analogues do not interact with AgaB at millimolar concentrations. The AgaB-DNJ@Fe3O4 enzyme-effector complex could be magnetically recycled and still showed a higher activity compared to free AgaB after four catalytic cycles. The “boost and recycle” procedure may provide interesting perspectives in glycosidase biocatalysis.
2 publications
Forato, Florian; Liu, Hao; Benoit, Roland; Fayon, Franck; Charlier, Cathy; Fateh, Amina; Defontaine, Alain; Tellier, Charles; Talham, Daniel R; Queffélec, Clémence; Bujoli, Bruno
Comparison of Zirconium Phosphonate-Modified Surfaces for Immobilizing Phosphopeptides and Phosphate-Tagged Proteins Article de journal
Dans: Langmuir, vol. 32, no. 22, p. 5480–5490, 2016, ISSN: 15205827.
@article{Forato2016,
title = {Comparison of Zirconium Phosphonate-Modified Surfaces for Immobilizing Phosphopeptides and Phosphate-Tagged Proteins},
author = {Florian Forato and Hao Liu and Roland Benoit and Franck Fayon and Cathy Charlier and Amina Fateh and Alain Defontaine and Charles Tellier and Daniel R Talham and Clémence Queffélec and Bruno Bujoli},
doi = {10.1021/acs.langmuir.6b01020},
issn = {15205827},
year = {2016},
date = {2016-01-01},
journal = {Langmuir},
volume = {32},
number = {22},
pages = {5480--5490},
abstract = {Different routes for preparing zirconium phosphonate-modified surfaces for immobilizing biomolecular probes are compared. Two chemical-modification approaches were explored to form self-assembled monolayers on commercially available primary amine-functionalized slides, and the resulting surfaces were compared to well-characterized zirconium phosphonate monolayer-modified supports prepared using Langmuir-Blodgett methods. When using POCl3 as the amine phosphorylating agent followed by treatment with zirconyl chloride, the result was not a zirconium-phosphonate monolayer, as commonly assumed in the literature, but rather the process gives adsorbed zirconium oxide/hydroxide species and to a lower extent adsorbed zirconium phosphate and/or phosphonate. Reactions giving rise to these products were modeled in homogeneous-phase studies. Nevertheless, each of the three modified surfaces effectively immobilized phosphopeptides and phosphopeptide tags fused to an affinity protein. Unexpectedly, the zirconium oxide/hydroxide modified surface, formed by treating the amine-coated slides with POCl3/Zr4+, afforded better immobilization of the peptides and proteins and efficient capture of their targets.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Different routes for preparing zirconium phosphonate-modified surfaces for immobilizing biomolecular probes are compared. Two chemical-modification approaches were explored to form self-assembled monolayers on commercially available primary amine-functionalized slides, and the resulting surfaces were compared to well-characterized zirconium phosphonate monolayer-modified supports prepared using Langmuir-Blodgett methods. When using POCl3 as the amine phosphorylating agent followed by treatment with zirconyl chloride, the result was not a zirconium-phosphonate monolayer, as commonly assumed in the literature, but rather the process gives adsorbed zirconium oxide/hydroxide species and to a lower extent adsorbed zirconium phosphate and/or phosphonate. Reactions giving rise to these products were modeled in homogeneous-phase studies. Nevertheless, each of the three modified surfaces effectively immobilized phosphopeptides and phosphopeptide tags fused to an affinity protein. Unexpectedly, the zirconium oxide/hydroxide modified surface, formed by treating the amine-coated slides with POCl3/Zr4+, afforded better immobilization of the peptides and proteins and efficient capture of their targets.
Goux, Marine; Fateh, Amina; Defontaine, Alain; Cinier, Mathieu; Tellier, Charles
In vivo phosphorylation of a peptide tag for protein purification Article de journal
Dans: Biotechnology Letters, vol. 38, no. 5, p. 767–772, 2016, ISSN: 1573-6776.
@article{Goux2016,
title = {In vivo phosphorylation of a peptide tag for protein purification},
author = {Marine Goux and Amina Fateh and Alain Defontaine and Mathieu Cinier and Charles Tellier},
url = {https://doi.org/10.1007/s10529-016-2040-4},
doi = {10.1007/s10529-016-2040-4},
issn = {1573-6776},
year = {2016},
date = {2016-01-01},
journal = {Biotechnology Letters},
volume = {38},
number = {5},
pages = {767--772},
abstract = {To design a new system for the in vivo phosphorylation of proteins in Escherichia coli using the co-expression of the α-subunit of casein kinase II (CKIIα) and a target protein, (Nanofitin) fused with a phosphorylatable tag.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
To design a new system for the in vivo phosphorylation of proteins in Escherichia coli using the co-expression of the α-subunit of casein kinase II (CKIIα) and a target protein, (Nanofitin) fused with a phosphorylatable tag.
6 publications
Cabezas, Yari; Legentil, Laurent; Robert-Gangneux, Florence; Daligault, Franck; Belaz, Sorya; Nugier-Chauvin, Caroline; Tranchimand, Sylvain; Tellier, Charles; Gangneux, Jean Pierre; Ferrières, Vincent
Leishmania cell wall as a potent target for antiparasitic drugs. A focus on the glycoconjugates Article de journal
Dans: Organic and Biomolecular Chemistry, vol. 13, no. 31, p. 8393–8404, 2015, ISSN: 14770520.
@article{Cabezas2015,
title = {Leishmania cell wall as a potent target for antiparasitic drugs. A focus on the glycoconjugates},
author = {Yari Cabezas and Laurent Legentil and Florence Robert-Gangneux and Franck Daligault and Sorya Belaz and Caroline Nugier-Chauvin and Sylvain Tranchimand and Charles Tellier and Jean Pierre Gangneux and Vincent Ferri{è}res},
doi = {10.1039/c5ob00563a},
issn = {14770520},
year = {2015},
date = {2015-01-01},
journal = {Organic and Biomolecular Chemistry},
volume = {13},
number = {31},
pages = {8393--8404},
publisher = {Royal Society of Chemistry},
abstract = {Although leishmaniasis has been studied for over a century, the fight against cutaneous, mucocutaneous and visceral forms of the disease remains a hot topic. This review refers to the parasitic cell wall and more particularly to the constitutive glycoconjugates. The structures of the main glycolipids and glycoproteins, which are species-dependent, are described. The focus is on the disturbance of the lipid membrane by existing drugs and possible new ones, in order to develop future therapeutic agents.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Although leishmaniasis has been studied for over a century, the fight against cutaneous, mucocutaneous and visceral forms of the disease remains a hot topic. This review refers to the parasitic cell wall and more particularly to the constitutive glycoconjugates. The structures of the main glycolipids and glycoproteins, which are species-dependent, are described. The focus is on the disturbance of the lipid membrane by existing drugs and possible new ones, in order to develop future therapeutic agents.
Brissonnet, Yoan; Ladevèze, Simon; Teze, David; Fabre, Emeline; Deniaud, David; Daligault, Franck; Tellier, Charles; Šesták, Sergej; Remaud-Simeon, Magali; Potocki-Veronese, Gabrielle; Gouin, Sébastien G
Polymeric iminosugars improve the activity of carbohydrate-processing enzymes Article de journal
Dans: Bioconjugate Chemistry, vol. 26, no. 4, p. 766–772, 2015, ISSN: 15204812.
@article{Brissonnet2015,
title = {Polymeric iminosugars improve the activity of carbohydrate-processing enzymes},
author = {Yoan Brissonnet and Simon Ladevèze and David Teze and Emeline Fabre and David Deniaud and Franck Daligault and Charles Tellier and Sergej Šesták and Magali Remaud-Simeon and Gabrielle Potocki-Veronese and Sébastien G Gouin},
doi = {10.1021/acs.bioconjchem.5b00081},
issn = {15204812},
year = {2015},
date = {2015-01-01},
journal = {Bioconjugate Chemistry},
volume = {26},
number = {4},
pages = {766--772},
abstract = {Multivalent iminosugars have recently emerged as powerful tools to inhibit the activities of specific glycosidases. In this work, biocompatible dextrans were coated with iminosugars to form linear and ramified polymers with unprecedently high valencies (from 20 to 900) to probe the evolution of the multivalent inhibition as a function of ligand valency. This study led to the discovery that polyvalent iminosugars can also significantly enhance, not only inhibit, the enzymatic activity of specific glycoside-hydrolase, as observed on two galactosidases, a fucosidase, and a bacterial mannoside phosphorylase for which an impressive 70-fold activation was even reached. The concept of glycosidase activation is largely unexplored, with a unique recent example of small-molecules activators of a bacterial O-GlcNAc hydrolase. The possibility of using these polymers as "artificial enzyme effectors may therefore open up new perspectives in therapeutics and biocatalysis.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Multivalent iminosugars have recently emerged as powerful tools to inhibit the activities of specific glycosidases. In this work, biocompatible dextrans were coated with iminosugars to form linear and ramified polymers with unprecedently high valencies (from 20 to 900) to probe the evolution of the multivalent inhibition as a function of ligand valency. This study led to the discovery that polyvalent iminosugars can also significantly enhance, not only inhibit, the enzymatic activity of specific glycoside-hydrolase, as observed on two galactosidases, a fucosidase, and a bacterial mannoside phosphorylase for which an impressive 70-fold activation was even reached. The concept of glycosidase activation is largely unexplored, with a unique recent example of small-molecules activators of a bacterial O-GlcNAc hydrolase. The possibility of using these polymers as "artificial enzyme effectors may therefore open up new perspectives in therapeutics and biocatalysis.
Arab-Jaziri, Faten; Bissaro, Bastien; Tellier, Charles; Dion, Michel; Fauré, Régis; O'Donohue, Michael J
Enhancing the chemoenzymatic synthesis of arabinosylated xylo-oligosaccharides by GH51 α-l-arabinofuranosidase Article de journal
Dans: Carbohydrate Research, vol. 401, p. 64–72, 2015, ISSN: 1873426X.
@article{Arab-Jaziri2015,
title = {Enhancing the chemoenzymatic synthesis of arabinosylated xylo-oligosaccharides by GH51 α-l-arabinofuranosidase},
author = {Faten Arab-Jaziri and Bastien Bissaro and Charles Tellier and Michel Dion and Régis Fauré and Michael J O'Donohue},
doi = {10.1016/j.carres.2014.10.029},
issn = {1873426X},
year = {2015},
date = {2015-01-01},
journal = {Carbohydrate Research},
volume = {401},
pages = {64--72},
publisher = {Elsevier Ltd},
abstract = {Random mutagenesis was performed on the α-l-arabinofuranosidase of Thermobacillus xylanilyticus in order to enhance its ability to perform transarabinofuranosylation using natural xylo-oligosaccharides as acceptors. To achieve this goal, a two-step, high-throughput digital imaging protocol involving a colorimetric substrate was used to screen a library of 30,000 mutants. In the first step this screen selected for hydrolytically-impaired mutants, and in the second step the screen identified mutants whose global activity was improved in the presence of a xylo-oligosaccharide mixture. Thereby, 199 mutants displaying lowered hydrolytic activity and modified properties were detected. In the presence of these xylo-oligosaccharides, most of the 199 (i.e., 70%) enzymes were less inhibited and some (18) mutants displayed an unambiguous alleviation of inhibition (textless25% loss of activity). More precise monitoring of reactions catalyzed by the most promising mutants revealed a significant improvement of the synthesis yields of transglycosylation products (up to 18% compared to 9% for the parental enzyme) when xylobiose was present in the reaction. Genetic analysis of improved mutants revealed that many of the amino acid substitutions that correlate with the modified phenotype are located in the vicinity of the active site, particularly in subsite -1. Consequently, we hypothesize that these mutations modify the active site topology or the molecular interaction network of the l-arabinofuranoside donor substrate, thus impairing the hydrolysis and concomitantly favoring transglycosylation onto natural acceptors.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Random mutagenesis was performed on the α-l-arabinofuranosidase of Thermobacillus xylanilyticus in order to enhance its ability to perform transarabinofuranosylation using natural xylo-oligosaccharides as acceptors. To achieve this goal, a two-step, high-throughput digital imaging protocol involving a colorimetric substrate was used to screen a library of 30,000 mutants. In the first step this screen selected for hydrolytically-impaired mutants, and in the second step the screen identified mutants whose global activity was improved in the presence of a xylo-oligosaccharide mixture. Thereby, 199 mutants displaying lowered hydrolytic activity and modified properties were detected. In the presence of these xylo-oligosaccharides, most of the 199 (i.e., 70%) enzymes were less inhibited and some (18) mutants displayed an unambiguous alleviation of inhibition (textless25% loss of activity). More precise monitoring of reactions catalyzed by the most promising mutants revealed a significant improvement of the synthesis yields of transglycosylation products (up to 18% compared to 9% for the parental enzyme) when xylobiose was present in the reaction. Genetic analysis of improved mutants revealed that many of the amino acid substitutions that correlate with the modified phenotype are located in the vicinity of the active site, particularly in subsite -1. Consequently, we hypothesize that these mutations modify the active site topology or the molecular interaction network of the l-arabinofuranoside donor substrate, thus impairing the hydrolysis and concomitantly favoring transglycosylation onto natural acceptors.
Teze, David; Daligault, Franck; Ferrières, Vincent; Sanejouand, Yves-Henri; Tellier, Charles
Semi-rational approach for converting a GH36 α-glycosidase into an α-transglycosidase Article de journal
Dans: Glycobiology, vol. 25, no. 4, p. 420–427, 2015, ISSN: 14602423.
@article{Teze2015,
title = {Semi-rational approach for converting a GH36 α-glycosidase into an α-transglycosidase},
author = {David Teze and Franck Daligault and Vincent Ferrières and Yves-Henri Sanejouand and Charles Tellier},
doi = {10.1093/glycob/cwu124},
issn = {14602423},
year = {2015},
date = {2015-01-01},
journal = {Glycobiology},
volume = {25},
number = {4},
pages = {420--427},
abstract = {A large number of retaining glycosidases catalyze both hydrolysis and transglycosylation reactions. In order to use them as catalysts for oligosaccharide synthesis, the balance between these two competing reactions has to be shifted toward transglycosylation. We previously designed a semi-rational approach to convert the Thermus thermophilus β-glycosidases into transglycosidases by mutating highly conserved residues located around the -1 subsite. In an attempt to verify that this strategy could be a generic approach to turn glycosidases into transglycosidases, Geobacillus stearothermophilus α-galactosidase (AgaB) was selected in order to obtain α-transgalactosidases. This is of particular interest as, to date, there are no efficient α-galactosynthases, despite the considerable importance of α-galactooligosaccharides. Thus, by site-directed mutagenesis on 14 AgaB residues, 26 single mutants and 22 double mutants were created and screened, of which 11 single mutants and 6 double mutants exhibited improved synthetic activity, producing 4-nitrophenyl α-d-galactopyranosyl-(1,6)-α-d-galactopyranoside in 26-57% yields against only 22% when native AgaB was used. It is interesting to note that the best variant was obtained by mutating a second-shell residue, with no direct interaction with the substrate or a catalytic amino acid. As this approach has proved to be efficient with both α- and β-glycosidases, it is a promising route to convert retaining glycosidases into transglycosidases.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
A large number of retaining glycosidases catalyze both hydrolysis and transglycosylation reactions. In order to use them as catalysts for oligosaccharide synthesis, the balance between these two competing reactions has to be shifted toward transglycosylation. We previously designed a semi-rational approach to convert the Thermus thermophilus β-glycosidases into transglycosidases by mutating highly conserved residues located around the -1 subsite. In an attempt to verify that this strategy could be a generic approach to turn glycosidases into transglycosidases, Geobacillus stearothermophilus α-galactosidase (AgaB) was selected in order to obtain α-transgalactosidases. This is of particular interest as, to date, there are no efficient α-galactosynthases, despite the considerable importance of α-galactooligosaccharides. Thus, by site-directed mutagenesis on 14 AgaB residues, 26 single mutants and 22 double mutants were created and screened, of which 11 single mutants and 6 double mutants exhibited improved synthetic activity, producing 4-nitrophenyl α-d-galactopyranosyl-(1,6)-α-d-galactopyranoside in 26-57% yields against only 22% when native AgaB was used. It is interesting to note that the best variant was obtained by mutating a second-shell residue, with no direct interaction with the substrate or a catalytic amino acid. As this approach has proved to be efficient with both α- and β-glycosidases, it is a promising route to convert retaining glycosidases into transglycosidases.
Saumonneau, Amélie; Champion, Elise; Peltier-Pain, Pauline; Molnar-Gabor, Dora; Hendrickx, Johann; Tran, Vinh; Hederos, Markus; Dekany, Gyula; Tellier, Charles
Design of an α-l-transfucosidase for the synthesis of fucosylated HMOs Article de journal
Dans: Glycobiology, vol. 26, no. 3, p. 261–269, 2015, ISSN: 14602423.
@article{Saumonneau2015a,
title = {Design of an α-l-transfucosidase for the synthesis of fucosylated HMOs},
author = {Amélie Saumonneau and Elise Champion and Pauline Peltier-Pain and Dora Molnar-Gabor and Johann Hendrickx and Vinh Tran and Markus Hederos and Gyula Dekany and Charles Tellier},
doi = {10.1093/glycob/cwv099},
issn = {14602423},
year = {2015},
date = {2015-01-01},
journal = {Glycobiology},
volume = {26},
number = {3},
pages = {261--269},
abstract = {Human milk oligosaccharides (HMOs) are recognized as benefiting breast-fed infants in multiple ways. As a result, there is growing interest in the synthesis of HMOs mimicking their natural diversity. Most HMOs are fucosylated oligosaccharides. α-l-Fucosidases catalyze the hydrolysis of α-l-fucose from the non-reducing end of a glucan. They fall into the glycoside hydrolase GH29 and GH95 families. The GH29 family fucosidases display a classic retaining mechanism and are good candidates for transfucosidase activity. We recently demonstrated that the α-l-fucosidase from Thermotoga maritima (TmαFuc) from the GH29 family can be evolved into an efficient transfucosidase by directed evolution (Osanjo et al. 2007). In this work, we developed semi-rational approaches to design an α-l-transfucosidase starting with the α-l-fucosidase from commensal bacteria Bifidobacterium longum subsp. infantis (BiAfcB, Blon-2336). Efficient fucosylation was obtained with enzyme mutants (L321P-BiAfcB and F34I/L321P-BiAfcB) enabling in vitro synthesis of lactodifucotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose III and lacto-N-difucohexaose I. The enzymes also generated more complex HMOs like fucosylated para-lacto-N-neohexaose (F-p-LNnH) and mono- or difucosylated lacto-N-neohexaose (F-LNnH-I, F-LNnH-II and DF-LNnH). It is worth noting that mutation at these two positions did not result in a strong decrease in the overall activity of the enzyme, which makes these variants interesting candidates for large-scale transfucosylation reactions. For the first time, this work provides an efficient enzymatic method to synthesize the majority of fucosylated HMOs.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Human milk oligosaccharides (HMOs) are recognized as benefiting breast-fed infants in multiple ways. As a result, there is growing interest in the synthesis of HMOs mimicking their natural diversity. Most HMOs are fucosylated oligosaccharides. α-l-Fucosidases catalyze the hydrolysis of α-l-fucose from the non-reducing end of a glucan. They fall into the glycoside hydrolase GH29 and GH95 families. The GH29 family fucosidases display a classic retaining mechanism and are good candidates for transfucosidase activity. We recently demonstrated that the α-l-fucosidase from Thermotoga maritima (TmαFuc) from the GH29 family can be evolved into an efficient transfucosidase by directed evolution (Osanjo et al. 2007). In this work, we developed semi-rational approaches to design an α-l-transfucosidase starting with the α-l-fucosidase from commensal bacteria Bifidobacterium longum subsp. infantis (BiAfcB, Blon-2336). Efficient fucosylation was obtained with enzyme mutants (L321P-BiAfcB and F34I/L321P-BiAfcB) enabling in vitro synthesis of lactodifucotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose III and lacto-N-difucohexaose I. The enzymes also generated more complex HMOs like fucosylated para-lacto-N-neohexaose (F-p-LNnH) and mono- or difucosylated lacto-N-neohexaose (F-LNnH-I, F-LNnH-II and DF-LNnH). It is worth noting that mutation at these two positions did not result in a strong decrease in the overall activity of the enzyme, which makes these variants interesting candidates for large-scale transfucosylation reactions. For the first time, this work provides an efficient enzymatic method to synthesize the majority of fucosylated HMOs.
André-Miral, Corinne; Koné, Fankroma Mt; Solleux, Claude; Grandjean, Cyrille; Dion, Michel; Tran, Vinh; Tellier, Charles
De novo design of a trans-β-N-acetylglucosaminidase activity from a GH1 β-glycosidase by mechanism engineering Article de journal
Dans: Glycobiology, vol. 25, no. 4, p. 394–402, 2015, ISSN: 14602423.
@article{Andre-Miral2015,
title = {De novo design of a trans-β-N-acetylglucosaminidase activity from a GH1 β-glycosidase by mechanism engineering},
author = {Corinne André-Miral and Fankroma Mt Koné and Claude Solleux and Cyrille Grandjean and Michel Dion and Vinh Tran and Charles Tellier},
doi = {10.1093/glycob/cwu121},
issn = {14602423},
year = {2015},
date = {2015-01-01},
journal = {Glycobiology},
volume = {25},
number = {4},
pages = {394--402},
abstract = {Glycoside hydrolases are particularly abundant in all areas of metabolism as they are involved in the degradation of natural polysaccharides and glycoconjugates. These enzymes are classified into 133 families (CAZy server, http://www.cazy.org) in which members of each family have a similar structure and catalytic mechanism. In order to understand better the structure/function relationships of these enzymes and their evolution and to develop new robust evolved glycosidases, we undertook to convert a Family 1 thermostable β-glycosidase into an exo-β-N-acetylglucosaminidase. This latter activity is totally absent in Family 1, while natural β-hexosaminidases belong to CAZy Families 3, 20 and 84. Using molecular modeling, we first showed that the docking of N-acetyl-d-glucosamine in the subsite -1 of the β-glycosidase from Thermus thermophilus (TtβGly) suggested several steric conflicts with active site amino-acids (N163, E338) induced by the N-acetyl group. Both N163A and N163D-E338G mutations induced significant N-acetylglucosaminidase activity in TtβGly. The double mutant N163D-E338G was also active on the bicyclic oxazoline substrate, suggesting that this mutated enzyme uses a catalytic mechanism involving a substrate-assisted catalysis with a noncovalent oxazoline intermediate, similar to the N-acetylglucosaminidases from Families 20 and 84. Furthermore, a very efficient trans-N-acetylglucosaminidase activity was observed when the double mutant was incubated in the presence of NAG-oxazoline as a donor and N-methyl-O-benzyl-N-(β-d-glucopyranosyl)-hydroxylamine as an acceptor. More generally, this work demonstrates that it is possible to exchange the specificities and catalytic mechanisms with minimal changes between phylogenetically distant protein structures.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Glycoside hydrolases are particularly abundant in all areas of metabolism as they are involved in the degradation of natural polysaccharides and glycoconjugates. These enzymes are classified into 133 families (CAZy server, http://www.cazy.org) in which members of each family have a similar structure and catalytic mechanism. In order to understand better the structure/function relationships of these enzymes and their evolution and to develop new robust evolved glycosidases, we undertook to convert a Family 1 thermostable β-glycosidase into an exo-β-N-acetylglucosaminidase. This latter activity is totally absent in Family 1, while natural β-hexosaminidases belong to CAZy Families 3, 20 and 84. Using molecular modeling, we first showed that the docking of N-acetyl-d-glucosamine in the subsite -1 of the β-glycosidase from Thermus thermophilus (TtβGly) suggested several steric conflicts with active site amino-acids (N163, E338) induced by the N-acetyl group. Both N163A and N163D-E338G mutations induced significant N-acetylglucosaminidase activity in TtβGly. The double mutant N163D-E338G was also active on the bicyclic oxazoline substrate, suggesting that this mutated enzyme uses a catalytic mechanism involving a substrate-assisted catalysis with a noncovalent oxazoline intermediate, similar to the N-acetylglucosaminidases from Families 20 and 84. Furthermore, a very efficient trans-N-acetylglucosaminidase activity was observed when the double mutant was incubated in the presence of NAG-oxazoline as a donor and N-methyl-O-benzyl-N-(β-d-glucopyranosyl)-hydroxylamine as an acceptor. More generally, this work demonstrates that it is possible to exchange the specificities and catalytic mechanisms with minimal changes between phylogenetically distant protein structures.
2014
Teze, David; Hendrickx, Johann; Czjzek, Mirjam; Ropartz, David; Sanejouand, Yves-Henri; Tran, Vinh; Tellier, Charles; Dion, Michel
Semi-rational approach for converting a GH1 ß-glycosidase into a ß-transglycosidase Article de journal
Dans: Protein Engineering, Design & Selection, vol. 27, no. 1, p. 13–19, 2014.
@article{teze2014semi,
title = {Semi-rational approach for converting a GH1 ß-glycosidase into a ß-transglycosidase},
author = {David Teze and Johann Hendrickx and Mirjam Czjzek and David Ropartz and Yves-Henri Sanejouand and Vinh Tran and Charles Tellier and Michel Dion},
doi = {10.1093/protein/gzt057},
year = {2014},
date = {2014-01-01},
urldate = {2014-01-01},
journal = {Protein Engineering, Design & Selection},
volume = {27},
number = {1},
pages = {13--19},
publisher = {Oxford University Press},
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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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.