An additional aromatic interaction improves the thermostability and thermophilicity of a mesophilic family 11 xylanase: Structural basis and molecular study

In a general approach to the understanding of protein adaptation to high te mperature, molecular models of the closely related mesophilic Streptomyces sp. S38 Xy11 and thermophilic Thermomonospora fusca TfxA family 11 xylanase s were built and compared with the three-dimensional (3D) structures of hom ologous enzymes. Some of the structural features identified as potential co ntributors to the higher thermostability of TfxA were introduced in Xy11 by site-directed mutagenesis in an attempt to improve its thermostability and thermophilicity. A new Y11-Y16 aromatic interaction, similar to that prese nt in TfxA and created in Xy11 by the T11Y mutation, improved both the ther mophilicity and thermostability. Indeed, the optimum activity temperature ( 70 vs. 60 degrees C) and the apparent T-m were increased by about 9 degrees C, and the mutant was sixfold more stable at 57 degrees C. The combined mu tations A82R/F168H/N169D/Delta 170 potentially creating a R82-D169 salt bri dge homologous to that present in TfxA improved the thermostability but not the thermophilicity. Mutations R82/D170 and S33P seemed to be slightly des tabilizing and devoid of influence on the optimal activity temperature of X y11. Structural analysis revealed that residues Y11 and Y16 were located on beta-strands B1 and B2, respectively. This interaction should increase the stability of the N-terminal part of Xy11. Moreover, Y11 and Y16 seem to fo rm an aromatic continuum with five other residues forming putative subsites involved in the binding of xylan (+3, +2, +1, -1, -2). Y11 and Y16 might r epresent two additional binding subsites (-3, -4) and the T11Y mutation cou ld thus improve substrate binding to the enzyme at higher temperature and t hus the thermophilicity of Xy11.