Functional roles of H98 and W99 and beta 2 alpha 2 loop dynamics in the alpha-L-arabinofuranosidase from Thermobacillus xylanilyticus

• Published in: The FEBS journal Vol. 279; no. 19; pp. 3598 - 3611
• Main Authors: Arab, Faten, Bissaro, Bastien, Barbe, Sophie, Saurel, Olivier, Debat, Helene, Dumon, Claire, Gervais, Virginie, Milon, Alain, André, Isabelle, Fauré, Régis, O'Donohue, Michael
• Format: Journal Article
• Language: English
• Published: Wiley 01.10.2012
• Subjects: Life Sciences; STD-NMR; glycoside hydrolase; ss a loop; a-l-arabinofuranosidase; molecular dynamics
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2012.08720.x

This study is focused on the elucidation of the functional role of the mobile beta 2a2 loop in the a-l-arabinofuranosidase from Thermobacillus xylanilyticus, and particularly on the roles of loop residues H98 and W99. Using site-directed mutagenesis, coupled to characterization methods including isothermal titration calorimetry (ITC) and saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy, and molecular dynamics simulations, it has been possible to provide a molecular level view of interactions and the consequences of mutations. Binding of para-nitrophenyl a-l-arabinofuranoside (pNP-a-l-Araf) to the wild-type arabinofuranosidase was characterized by Kd values (0.32 and 0.16 mm, from ITC and STD-NMR respectively) that highly resembled that of the arabinoxylo-oligosaccharide XA3XX (0.21 mm), and determination of the thermodynamic parameters of enzyme : pNP-a-l-Araf binding revealed that this process is driven by favourable entropy, which is linked to the movement of the beta 2a2 loop. Loop closure relocates the solvent-exposed W99 into a buried location, allowing its involvement in substrate binding and in the formation of a functional active site. Similarly, the data underline the role of H98 in the dynamic formation and definition of a catalytically operational active site, which may be a specific feature of a subset of GH51 arabinofuranosidases. Substitution of H98 and W99 by alanine or phenylalanine revealed that mutations affected KM and/or kcat. Molecular dynamics performed on W99A implied that this mutation causes the loss of a hydrogen bond and leads to an alternative binding mode that is detrimental for catalysis. STD-NMR experiments revealed altered binding of the aglycon motif in the active site, combined with reduced STD intensities of the a-l-arabinofuranosyl moiety for W99 substitutions.