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

Abstract

In a general approach to the understanding of protein adaptation to high temperature, molecular models of the closely related mesophilic Streptomyces sp. S38 Xyl1 and thermophilic Thermomonospora fusca TfxA family 11 xylanases were built and compared with the three-dimensional~3D! structures of homologous enzymes. Some of the structural features identified as potential contributors to the higher thermostability of TfxA were introduced in Xyl1 by site-directed mutagenesis in an attempt to improve its thermostability and thermophilicity. A new Y11-Y16 aromatic interaction, similar to that present in TfxA and created in Xyl1 by the T11Y mutation, improved both the thermophilicity and thermostability. Indeed, the optimum activity temperature~70 vs. 60 8C! and the apparent T m were increased by about 9 8C, and the mutant was sixfold more stable at 57 8C. The combined mutations A82R0F168H0N169D0D170 potentially creating a R82-D169 salt bridge homologous to that present in TfxA improved the thermostability but not the thermophilicity. Mutations R820D170 and S33P seemed to be slightly destabilizing and devoid of influence on the optimal activity temperature of Xyl1. Structural analysis revealed that residues Y11 and Y16 were located on b-strands B1 and B2, respectively. This interaction should increase the stability of the N-terminal part of Xyl1. Moreover, Y11 and Y16 seem to form an aromatic continuum with five other residues forming putative subsites involved in the binding of xylañ ϩ3, ϩ2, ϩ1, Ϫ1, Ϫ2!. Y11 and Y16 might represent two additional binding subsites~Ϫ3, Ϫ4! and the T11Y mutation could thus improve substrate binding to the enzyme at higher temperature and thus the thermophilicity of Xyl1.

Engineering the Pattern of Protein Glycosylation Modulates the Thermostability of a GH11 Xylanase

“…Endo-␤-1,4-xylanase A (XynA) is a mesostable GH11 glycosyl hydrolase secreted by Bacillus subtilis, and protein engineering studies have identified multiple factors that contribute to thermostabilization of the protein, including optimization of hydrogen bonds (34), hydrophobic interactions (35), optimization of the electrostatic surface of proteins (36), and the introduction of disulfide bonds (37). Here we present a novel approach to improve protein thermostability by rational design, in which the XynA expressed in P. pastoris has been used to investigate the effect of glycosylation on protein stability.…”

Nanopeptides: Non - Covalent Interactions in Chemistry and Biological Functions

“…In 1985, Burley and Petsko observed, "that on an average about 60% of aromatic side chains in proteins are involved in aromatic pairs, 80% of which form networks of three or more interacting aromatic side chains [1][2][3][4][5][6]. Phenyl ring centroids are separated by a preferential distance of between 4.5 Å and 7 Å, and dihedral angles approaching 90° are most common"…”

Association of Novel Domain in Active Site of Archaic Hyperthermophilic Maltogenic Amylase from Staphylothermus marinus

“…Amino acid analysis showed that SMMA has a significantly higher frequency of Ile (74, 10.6%) than do the other four enzymes (average 24, 4.0%), but it has significantly lower Gln content (9, 1.3%). Ile occurs often in thermophilic proteins, and Gln is a labile amino acid that is deaminated at high temperatures, which suggests that known structural fac- tors involved in thermostability correlate well with the SMMA structure (43)(44)(45).…”