Functional roles of H98 and W99 and β2α2 loop dynamics in the α‐l‐arabinofuranosidase from Thermobacillus xylanilyticus

Abstract

This study is focused on the elucidation of the functional role of the mobile b2a2 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 K d values (0.32 and 0.16 mM, from ITC and STD-NMR respectively) that highly resembled that of the arabinoxylo-oligosaccharide XA 3 XX (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 b2a2 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 K M and ⁄ or k cat . 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.

Probing the determinants of the transglycosylation/hydrolysis partition in a retaining α-l-arabinofuranosidase

“…Generally, the loops surrounding the active site appear to be stabilized by the presence of the ligand, consistent with previous observations (Suppl. Figure S7) [63]. In comparison with wild-type enzyme, the MD simulation performed on F26L-R69H-N216W revealed only slight conformational differences in the vicinity of the active site.…”

A novel bifunctional GH51 exo-α-l-arabinofuranosidase/endo-xylanase from Alicyclobacillus sp. A4 with significant biomass-degrading capacity

“…Until now, bacterial GH51 Abfs from Anoxybacillus kestanbolensis [ 17 ], Clostridium stercorarium [ 18 ], Geobacillus stearothermophilus [ 19 ], Bacillus pumilus [ 20 ], Bifidobacterium longum [ 21 ], and Streptomyces spp. [ 22 – 24 ] have been characterized, and the structures of six bacterial Abfs have been resolved, which all perform hydrolysis via a retaining mechanism [ 25 ]. Previous studies indicate that GH51 Abfs have various modes of action on different substrates.…”

New Insights into the Role of T3 Loop in Determining Catalytic Efficiency of GH28 Endo-Polygalacturonases

“…In other words, the importance of the amino acid side chain at position 94 might be comparable to that of Ile116 at the tip of the thumb-loop of GH11 xylanase from Thermobacillus xylanilyticus [ 19 – 21 ], and that of W99 in the β2α2 loop region of GH51 arabinofuranosidase from T . xylanilyticus [ 22 ]. This is the first report on the role of T3 loop in the catalytic efficiency of GH28 endo-PG.…”

“…The functions of enzymes are intimately related to protein flexibility and conformation of some structural elements [ 15 , 16 ]. As the most flexible parts of protein structures [ 17 ], loop regions have been shown to involve the catalytic activity of enzymes and play major roles in substrate selectivity and recognition by facilitating substrate binding to the cleft [ 18 – 22 ]. However, the exact roles of the active site loops of GH28 endo-PGs are unknown yet.…”