Insight into the mechanism of thermostabilization of GH10 xylanase from Bacillus sp. strain TAR-1 by the mutation of S92 to E

Abstract

The mechanism of thermostabilization of GH10 xylanase, XynR, from Bacillus sp. strain TAR-1 by the mutation of S92 to E was investigated. Thermodynamic analysis revealed that thermostabilization was driven by the decrease in entropy change of activation for thermal inactivation. Crystallographic analysis suggested that this mutation suppressed the fluctuation of the amino acid residues at position 92-95.

Activity-stability trade-off observed in variants at position 315 of the GH10 xylanase XynR

“…It should be noted that the 4 peak regions with residues 15-20, 75-83, 105-109, and 264-267 are formed by the crystal packing effects. Therefore, this result shows that the largest deviation occurs in the residues 311-331, which contains the Ca 2+ binding site in the α helix (317-320) and the extended loop (321-331) [13].…”

“…The structure was refined at 1.90 Å resolution. The whole structure of T315Q exhibited no difference compared to WT [13] with rmsd of 0.65 Å for 353 C atoms. Figure 1A shows the mutation-site structure of T315Q.…”

“…Figure 6 shows the rmsd distance plot after superposition of the structures of T315Q (this study) and WT [13]. It should be noted that the 4 peak regions with residues 15-20, 75-83, 105-109, and 264-267 are formed by the crystal packing effects.…”

“…As shown in Table 1, the space group of the crystal was P21, and the structure was refined at 1.82 Å resolution. The whole structure of WT complexed with xylobiose exhibited no difference compared to ligand-free WT [13] with rmsd of 0.21 Å for 353 Cα atoms in chain A. Figure 7A shows the active-site structure of WT complexed with xylobiose, showing that in the region (residue 311-331) with the largest deviation between T315Q and WT (Fig.…”

“…The collected diffraction data were processed with XDS [17]. Molecular replacement was conducted using the structure of WT [13] as a search model (PDB, 7CPK). Structure refinement was conducted using COOT [18] and PHENIX [19].…”