title: Mutation of a pH-modulating residue in a GH51 α-l-arabinofuranosidase leads to a severe reduction of the secondary hydrolysis of transfuranosylation products.
abstract: 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 (kcat, KM, pKa 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 pKa 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
  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.
authors.affiliation: Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France.
authors.forename: Bastien
authors.initials: B
authors.lastname: Bissaro
authors.forename: Olivier
authors.initials: O
authors.lastname: Saurel
authors.forename: Faten
authors.initials: F
authors.lastname: Arab-Jaziri
authors.forename: Luc
authors.initials: L
authors.lastname: Saulnier
authors.forename: Alain
authors.initials: A
authors.lastname: Milon
authors.forename: Maija
authors.initials: M
authors.lastname: Tenkanen
authors.forename: Pierre
authors.initials: P
authors.lastname: Monsan
authors.forename: Michael J
authors.initials: MJ
authors.lastname: O'Donohue
authors.forename: Régis
authors.initials: R
authors.lastname: Fauré
mesh_terms: D001089:Arabinose / Q000378:metabolism*; D001406:Bacillaceae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D002855:Chromatography, Thin Layer; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D001089:Arabinose; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase
keywords: 4-nitrochatecol α-l-arabinofuranoside; 4NTC-α-l-Araf; AXOS; Abfs; FH; GH; KIE; Pentoses/furanoses; R(T); STD NMR; Saturation Transfer Difference Nuclear Magnetic Resonance; T/H ratio; Transglycosylation; TxAbf; TxAbf(ǂ); X; Y; arabinoxylo-oligosaccharides; d-Xylp; d-xylopyranosyl; donor conversion rate; furanoside hydrolase; glycoside hydrolase; inactivated form (E176A) of TxAbf; kinetic isotope effects; l-Araf; l-arabinofuranosyl; mNP; meta-nitrophenol; oNP; ortho-nitrophenol; pH-dependent inhibition; pK(a) modulation; pNP; pNP-α-l-Araf; para-nitrophenol; para-nitrophenyl α-l-arabinofuranoside; transfer rate; transglycosylation/hydrolysis ratio; yield; α-l-arabinofuranosidase from Thermobacillus xylanilyticus; α-l-arabinofuranosidases