부산대학교 도서관

This study is focused on the elucidation of the functional role of the mobile β2α2 loop in the α- 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 α- l-arabinofuranoside ( pNP-α- l-Ara f) to the wild-type arabinofuranosidase was characterized by Kd values (0.32 and 0.16 m m, from ITC and STD-NMR respectively) that highly resembled that of the arabinoxylo-oligosaccharide XA3XX (0.21 m m), and determination of the thermodynamic parameters of enzyme : pNP-α- l-Ara f binding revealed that this process is driven by favourable entropy, which is linked to the movement of the β2α2 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 α- l-arabinofuranosyl moiety for W99 substitutions. [ABSTRACT FROM AUTHOR]