Mutation of a pH-modulating residue in a GH51 α-L-arabinofuranosidase leads to a severe reduction of the secondary hydrolysis of transfuranosylation products ☆ Bastien Bissaro a,b,c , Olivier Saurel a,d , Faten Arab-Jaziri a,b,c , Luc Saulnier e , Alain Milon a,d , Maija Tenkanen f , Pierre Monsan a,b,c , Michael J. O'Donohue a,b,c , Régis Fauré a,b,c, ⁎ a Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France b INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France c CNRS, UMR5504, F-31400 Toulouse, France d CNRS, IPBS UMR 5089, Institut de Pharmacologie et de Biologie Structurale, 205 route de Narbonne, BP 64182 Toulouse, France e UR1268 Biopolymères Interactions Assemblages, INRA, 44300 Nantes, France f Department of Food and Environmental Sciences, Faculty of Agriculture and Forestry, University of Helsinki, P.O. Box 27, FI-00014, Finland abstract article info Article history: Received 10 June 2013 Received in revised form 23 September 2013 Accepted 4 October 2013 Available online 17 October 2013 Keywords: Pentoses/furanoses Transglycosylation pK a modulation pH-dependent inhibition STD NMR 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 α-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 (k cat , K M ,pK a 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 pK a 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 α-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. © 2013 Elsevier B.V. All rights reserved. 1. Introduction So far, very few studies have dealt with the development of furanoside hydrolases (FHs) as glycosynthetic tools, with most work being performed on hexose and/or pyranose-acting enzymes [2–5]. This is unsurprising since, not only are FHs a relatively small subgroup of glycoside hydrolases (GHs), but also furanose chemistry is not a mainstream topic, in part because furanosides are often tricky to handle. Nevertheless, furanoses are quite widespread in Nature, being found in the cell wall polysaccharides of most plants and those of a variety of pathogenic microorganisms. Therefore, the development of chemo- enzymatic tools and methods is a pertinent pursuit, notably in the context of the production of new furanose-based products for a whole Biochimica et Biophysica Acta 1840 (2014) 626–636 Abbreviations: Abfs, α-L-arabinofuranosidases; AXOS, arabinoxylo-oligosaccharides; D- Xylp, D-xylopyranosyl; FH, furanoside hydrolase; GH, glycoside hydrolase; KIE, kinetic isotope effects; L-Araf, L-arabinofuranosyl; 4NTC-α-L-Araf, 4-nitrochatecol α-L-arabinofuranoside; mNP, meta-nitrophenol; oNP, ortho-nitrophenol; pNP, para-nitrophenol; pNP-α-L-Araf, para-nitrophenyl α-L-arabinofuranoside; R T , transfer rate; STD NMR, Saturation Transfer Difference Nuclear Magnetic Resonance; T/H ratio, transglycosylation/hydrolysis ratio; TxAbf, α-L-arabinofuranosidase from Thermobacillus xylanilyticus; TxAbf E , inactivated form (E176A) of TxAbf; X, donor conversion rate; Y, yield ☆ The specific abbreviated names of different AXOS, such as A 3 X and XA 3 XX, were generated using the naming system developed by Fauré et al. [1]. ⁎ Corresponding author at: Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés, 135 Avenue de Rangueil, 31077 Toulouse cedex 4, France. Tel.: +33 5 6155 9488; fax: +33 5 6155 9400. E-mail address: regis.faure@insa-toulouse.fr (R. Fauré). 0304-4165/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbagen.2013.10.013 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen