期刊
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS
卷 1840, 期 1, 页码 626-636出版社
ELSEVIER
DOI: 10.1016/j.bbagen.2013.10.013
关键词
Pentoses/furanoses; Transglycosylation; pK(a)modulation; pH-dependent inhibition; STD NMR
资金
- Institut National de la Recherche Agronomique
- Region MidiPyrenees
- European Regional Development Fund
- SICOVAL
- Infrastructures en Biologie Sante et Agronomie (IBiSa, France)
- Centre National de la Recherche Scientifique (CNRS)
- Institut National de la Recherche Agronomique (INRA)
- French Research Ministry
- CNRS
- Universite Paul Sabatier
- Region Midi-Pyrenees
- European structural funds
Background: The development of enzyme-mediated glycosynthesis using glycoside hydrolases is still an inexact science, because the underlying molecular determinants of transglycosylation are not well understood. In the framework of this challenge, this study focused on the family GH51 a-L-arabinofuranosidase from Thermobacillus xylanilyticus, with the aim to understand why the mutation of position 344 provokes a significant modification of the transglycosylation/hydrolysis partition. Methods: Detailed kinetic analysis (kat, Km, plc determination and time-course NMR kinetics) and saturation transfer difference nuclear magnetic resonance spectroscopy was employed to determine the synthetic and hydrolytic ability modification induced by the redundant N344 mutation disclosed in libraries from directed evolution. Results: The mutants N344P and N344Y displayed crippled hydrolytic abilities, and thus procured improved transglycosylation yields. This behavior was correlated with an increased pK(a) of the catalytic nucleophile (E298), the pKa of the acid/base catalyst remaining unaffected. Finally, mutations at position 344 provoked a pH-dependent product inhibition phenomenon, which is likely to be the result of a significant modification of the proton sharing network in the mutants. Conclusions and general significance: Using a combination of biochemical and biophysical methods, we have studied TxAbf-N344 mutants, thus revealing some fundamental details concerning pH modulation. Although these results concern a GH51 a-L-arabinofuranosidase, it is likely that the general lessons that can be drawn from them will be applicable to other glycoside hydrolases. Moreover, the effects of mutations at position 344 on the transglycosylation/hydrolysis partition provide clues as to how TxAbf can be further engineered to obtain an efficient transfuranosidase. (C) 2013 Elsevier B.V. All rights reserved.
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