Probing the determinants of the transglycosylation/hydrolysis partition in a retaining α-L-arabinofuranosidase
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The use of retaining glycoside hydrolases as synthetic tools for glycochemistry is highly topical and the focus of considerable research. However, due to the incomplete identification of the molecular determinants of the transglycosylation/hydrolysis partition (T/H), rational engineering of retaining glycoside hydrolases to create transglycosylases remains challenging. Therefore, to understand better the factors that underpin transglycosylation in a GH51 retaining α-L-arabinofuranosidase from Thermobacillus xylanilyticus, the investigation of this enzyme's active site was pursued. Specifically, the properties of two mutants, F26L and L352M, located in the vicinity of the active site are described, using kinetic and 3D structural analyses and molecular dynamics simulations. The results reveal that the presence of L352M in the context of a triple mutant (also containing R69H and N216W) generates changes both in the donor and acceptor subsites, the latter being the result of a domino-like effect. Overall, the mutant R69H-N216W-L352M displays excellent transglycosylation activity (70 % yield, 78 % transfer rate and reduced secondary hydrolysis of the product). In the course of this study, the central role played by the conserved R69 residue was also reaffirmed. The mutation R69H affects both the catalytic nucleophile and the acid/base, including their flexibility, and has a determinant effect on the T/H partition. Finally, the results reveal that increased loop flexibility in the acceptor subsites creates new interactions with the acceptor, in particular with a hydrophobic binding platform composed of N216W, W248 and W302.
| Originalsprog | Engelsk |
|---|---|
| Tidsskrift | New Biotechnology |
| Vol/bind | 62 |
| Sider (fra-til) | 68-78 |
| Antal sider | 11 |
| ISSN | 1871-6784 |
| DOI | |
| Status | Udgivet - 25 maj 2021 |
Bibliografisk note
Funding Information:
The PhD fellowships of J. Zhao and B. Bissaro were supported by CSC (China Scholarship Council) and INRAE (Institut National de la Recherche pour l'Agriculture, l'Alimentation et l'Environnement) [CJS] respectively. This work was performed in the framework of the French Danish research collaboration Program (IFD, N? 15/2015/CSU.8.2.1). Travel to synchrotron facilities was supported by the Danish Ministry of Higher Education and Science through the Instrument Center DANSCATT.The NMR analyses were performed using facilities at MetaToul (Metabolomics & Fluxomics Facilities, Toulouse, France, www.metatoul.fr), which is part of the national infrastructure MetaboHUB (The French National infrastructure for metabolomics and fluxomics, www.metabohub.fr) and is supported by grants from the Re?gion Midi-Pyre?ne?es, the European Regional Development Fund, SICOVAL, IBiSa-France,CNRS, and INRAE. G. Lippens and N. Cox (TBI) are gratefully acknowledged for insightful discussions and technical development of the NMR pseudo-2D kinetics experiments. Parts of this research were performed on beamlines ID30-B and ID23-1 at the European Synchrotron Radiation Facility (ESRF, Grenoble, France). We are grateful for the assistance in using beamline ID30-B and ID23-1 respectively. Research was also performed at beamline I911-3 of MAX II (Lund, Sweden). Being able to access this beamline was highly appreciated. This work was granted access to the HPC resources on the Computing mesocenter of R?gion Midi-Pyr?n?es (CALMIP, Toulouse, France).
Funding Information:
The NMR analyses were performed using facilities at MetaToul (Metabolomics & Fluxomics Facilities, Toulouse, France, www.metatoul.fr ), which is part of the national infrastructure MetaboHUB (The French National infrastructure for metabolomics and fluxomics, www.metabohub.fr ) and is supported by grants from the Région Midi-Pyrénées, the European Regional Development Fund , SICOVAL , IBiSa-France, CNRS , and INRAE . G. Lippens and N. Cox (TBI) are gratefully acknowledged for insightful discussions and technical development of the NMR pseudo-2D kinetics experiments. Parts of this research were performed on beamlines ID30-B and ID23-1 at the European Synchrotron Radiation Facility (ESRF, Grenoble, France). We are grateful for the assistance in using beamline ID30-B and ID23-1 respectively. Research was also performed at beamline I911-3 of MAX II (Lund, Sweden). Being able to access this beamline was highly appreciated. This work was granted access to the HPC resources on the Computing mesocenter of Région Midi-Pyrénées (CALMIP, Toulouse, France).
Funding Information:
The PhD fellowships of J. Zhao and B. Bissaro were supported by CSC (China Scholarship Council) and INRAE (Institut National de la Recherche pour l’Agriculture, l’Alimentation et l’Environnement) [CJS] respectively. This work was performed in the framework of the French Danish research collaboration Program (IFD, N° 15/2015/CSU.8.2.1). Travel to synchrotron facilities was supported by the Danish Ministry of Higher Education and Science through the Instrument Center DANSCATT.
Publisher Copyright:
© 2021 Elsevier B.V.
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