Benoît DAVID
Doctorant Université
mai 2017
| Équipes : |
Publications
1 publication
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.
2 publications
David, Benoît
Conception rationnelle dénzyme: conversion de glycoside hydrolases en transglycosidases Thèse
Université de Nantes, 2017.
@phdthesis{david2017conception,
title = {Conception rationnelle dénzyme: conversion de glycoside hydrolases en transglycosidases},
author = {Benoît David},
url = {https://www.theses.fr/2017NANT1044},
year = {2017},
date = {2017-01-01},
school = {Université de Nantes},
abstract = {Catalyseurs de la dégradation de polysaccharides dans le cadre de diverses applications industrielles, de nombreuses glycoside hydrolases (GH) possèdent également une activité de transglycosylation qui peut être exploitée pour la synthèse dóligosaccharides. Afin dáugmenter cette activité, minoritaire par rapport à l'hydrolyse, des expériences de mutagenèse rationnelle peuvent être employées. Toutefois, lénsemble des bases moléculaires régissant l’équilibre entre ces deux activités reste en revanche difficile a élucider. L'étude de quatre GH (Ttβgly, AgaD, TcTS, TrSA) par simulation de dynamique moléculaire a permis la découverte de canaux déau internes à leurs structures et connectant le site actif au milieu. Cette observation suggère que les canaux déau internes aux GH pourraient être impliqués dans leur activité d'hydrolyse. Plusieurs paires de résidus bordant deux de ces canaux ont été mis en évidence chez Ttβgly et AgaD et semblent contrôler le passage de léau du canal vers le site actif. La mutagenèse de ces résidus a été entreprise afin de tenter dáugmenter láctivité de transglycosylation chez ces deux enzymes. Une réduction de l'hydrolyse d'un facteur 7 et 50 au profit de láctivité de transglycosylation a été caractérisée chez les deux meilleurs mutants de Ttβgly et AgaD, respectivement. Lánalyse des simulations a révélé que ces résultats étaient corrélés à une augmentation de la dynamique des molécules déau internes aux deux canaux étudiés. Cette étude souligne ainsi límportance fonctionnelle de léau interne aux hydrolases et suggère que língénierie de sa dynamique peut constituer une approche originale pour convertir les GH en transglycosidases.},
keywords = {},
pubstate = {published},
tppubtype = {phdthesis}
}
Catalyseurs de la dégradation de polysaccharides dans le cadre de diverses applications industrielles, de nombreuses glycoside hydrolases (GH) possèdent également une activité de transglycosylation qui peut être exploitée pour la synthèse dóligosaccharides. Afin dáugmenter cette activité, minoritaire par rapport à l'hydrolyse, des expériences de mutagenèse rationnelle peuvent être employées. Toutefois, lénsemble des bases moléculaires régissant l’équilibre entre ces deux activités reste en revanche difficile a élucider. L'étude de quatre GH (Ttβgly, AgaD, TcTS, TrSA) par simulation de dynamique moléculaire a permis la découverte de canaux déau internes à leurs structures et connectant le site actif au milieu. Cette observation suggère que les canaux déau internes aux GH pourraient être impliqués dans leur activité d'hydrolyse. Plusieurs paires de résidus bordant deux de ces canaux ont été mis en évidence chez Ttβgly et AgaD et semblent contrôler le passage de léau du canal vers le site actif. La mutagenèse de ces résidus a été entreprise afin de tenter dáugmenter láctivité de transglycosylation chez ces deux enzymes. Une réduction de l'hydrolyse d'un facteur 7 et 50 au profit de láctivité de transglycosylation a été caractérisée chez les deux meilleurs mutants de Ttβgly et AgaD, respectivement. Lánalyse des simulations a révélé que ces résultats étaient corrélés à une augmentation de la dynamique des molécules déau internes aux deux canaux étudiés. Cette étude souligne ainsi límportance fonctionnelle de léau interne aux hydrolases et suggère que língénierie de sa dynamique peut constituer une approche originale pour convertir les GH en transglycosidases.
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.