Crystallographic analysis shows substrate binding at the −3 to +1 active-site subsites and at the surface of glycoside hydrolase family 11 endo-1,4-β-xylanases

Abstract

GH 11 (glycoside hydrolase family 11) xylanases are predominant enzymes in the hydrolysis of heteroxylan, an abundant structural polysaccharide in the plant cell wall. To gain more insight into the protein-ligand interactions of the glycone as well as the aglycone subsites of these enzymes, catalytically incompetent mutants of the Bacillus subtilis and Aspergillus niger xylanases were crystallized, soaked with xylo-oligosaccharides and subjected to X-ray analysis. For both xylanases, there was clear density for xylose residues in the -1 and -2 subsites. In addition, for the B. subtilis xylanase, there was also density for xylose residues in the -3 and +1 subsite showing the spanning of the -1/+1 subsites. These results, together with the observation that some residues in the aglycone subsites clearly adopt a different conformation upon substrate binding, allowed us to identify the residues important for substrate binding in the aglycone subsites. In addition to substrate binding in the active site of the enzymes, the existence of an unproductive second ligand-binding site located on the surface of both the B. subtilis and A. niger xylanases was observed. This extra binding site may have a function similar to the separate carbohydrate-binding modules of other glycoside hydrolase families.

Isothermal titration calorimetry and surface plasmon resonance allow quantifying substrate binding to different binding sites of Bacillus subtilis xylanase

“…Both ITC and SPR experiments were performed in McIlvaine buffer (0.10 M citrate and 0.20 M disodium hydrogen phosphate, pH 6.0) at 25 °C. For the experiments, xylohexaose (X 6 ) was chosen as substrate in these experiments because (i) it can fill all six subsites in the AS of XBS, (ii) it is large enough to span all SBS subsites, and (iii) it is too short to bind both the AS and SBS simultaneously [5].…”

“…Therefore, three enzyme variants were produced, and their purity was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining analogous to previous descriptions [6,7]. The first one, XBS_E172A, was a catalytically incompetent mutant that can still bind substrate to the AS and SBS [5]. A two-site binding model was used to fit the data obtained with XBS_E172A.…”

“…In the study presented here, the potential of isothermal titration calorimetry (ITC) and SPR, two commonly used techniques quantifying binding, was explored for their ability to quantify the strength of substrate binding to the AS and SBS of the glycoside hydrolase family (GH)-11 Bacillus subtilis xylanase (XBS) separately. XBS differs only one residue from the B. circulans xylanase, and the SBSs of the two enzymes are identical [5]. The SBS plays an important role for XBS in binding and hydrolysis of polymeric substrates and in substrate targeting [6,7].…”

A novel In-situ Enzymatic Cleaning Method for Reducing Membrane Fouling in Membrane Bioreactors (MBRs)

“…The further characterics of the enzyme were given by references (Vandermarliere et al, 2008;Pollet, 2010 (1) Determination of the pH optimum…”

Probing the Role of Sigma π Interaction and Energetics in the Catalytic Efficiency of Endo-1,4-β-Xylanase

“…3). Y102, Y106, W108, Y125, and W167 were previously identified as critical for substrate binding and hydrolysis through crystallographic and mutagenesis studies (7,12,13,14,15). N74 is important for activity at the optimal pH, and I157 is involved in product release from the active site (1,8).…”