Characterization of active-site aromatic residues in xylanase A from Streptomyces lividans

Abstract

The role of four aromatic residues (W85, Y172, W266 and W274) in the structure-function relationship in xylanase A from Streptomyces lividans (XlnA) was investigated by site-directed mutagenesis where each residue was subjected to three substitutions (W85A/H/F; W266A/H/F; W274A/H/F and Y172A/F/S). These four amino acids are highly conserved among family 10 xylanases and structural data have implicated them in substrate binding at the active site. Far-UV circular dichroism spectroscopy was used to show that the overall structure of XlnA was not affected by any of these mutations. High-performance liquid chromatographic analysis of the hydrolysis products of birchwood xylan and xylopentaose showed that mutation of these aromatic residues did not alter the enzyme's mode of action. As expected, though, it did reduce the affinity of XlnA for birchwood xylan. A comparison of the kinetic parameters of different mutants at the same position demonstrated the importance of the aromatic nature of W85, Y172 and W274 in substrate binding. Replacement of these residues by a phenylalanine resulted in mutant proteins with a K(M) closer to that of the wild-type protein in comparison with the other mutations analyzed. The kinetic analysis of the mutant proteins at position W266 indicated that this amino acid is important for both substrate binding and efficient catalysis by XlnA. These studies also demonstrated the crucial role of these active site aromatic residues for the thermal stability of XlnA.

Novel intracellular GH10 xylanase from Cohnella laeviribosi HY-21: Biocatalytic properties and alterations of substrate specificities by site-directed mutagenesis of Trp residues

“…On the other hand, iXylC was completely inactivated by Trp residue-specific chemical modifiers such as 1 mM Hg 2+ and 5 mM N-bromosuccinimide, as had also been demonstrated in many other xylanases (Cheng et al, 2008;Li et al, 2008). These results correspond to the demonstration that some Trp residues in the active site of extracellular GH10 xylanases play a key role in catalysis and/or substrate-binding (Roberge et al, 1999;Zolotnitsky et al, 2004). The significant inhibition (>40% of original activity) of iXylC activity was also noted in the presence of 1 mM Cu 2+ , 5 mM EDTA, and 5 mM N-ethylmaleimide.…”

“…Functions of some aromatic amino acid residues, such as His and Trp, conserved in active site of extracellular GH10 xylanases have recently been identified to play an essential role in catalysis and/or substrate-binding of the enzymes by site-directed mutagenesis (Roberge et al, 1999) and mapping of GH substrate sub- sites (Zolotnitsky et al, 2004). However, no study has yet been reported on functional characterization of active site aromatic residues conserved in intracellular GH10 xylanases, which are expected to have distinct substrate specificity compared to extracellular GH10 enzymes.…”

Novel GH10 Xylanase, with a Fibronectin Type 3 Domain, from Cellulosimicrobium sp. Strain HY-13, a Bacterium in the Gut of Eisenia fetida

“…The rXylK1 was relatively unaffected by sulfhydryl reagents (5 mM) such as sodium azide, iodoacetamide, and N-ethylmaleimide, while the enzyme lost 68% of its original activity when preincubated with 5 mM EDTA for 10 min. The complete inhibition of rXylK1 by 5 mM N-bromosuccinimide was in good agreement with the fact that three Trp residues in the highly conserved region of the GH10 enzymes are critically involved in enzyme-substrate interaction, as shown for Streptomyces lividans (16) and Geobacillus stearothermophilus T-6 (18) GH10 xylanases. It was predicted that the three residues Trp118, Trp306, and Trp314 in premature XylK1 might be responsible for catalysis and substrate binding of the enzyme.…”

Sequencing and Expression of the Xylanase Gene 2 from <i>Trichoderma reesei</i> Rut C-30 and Characterization of the Recombinant Enzyme and Its Activity on Xylan

“…Unlike Family 10 xylanases, the native Xyn2 protein has no stabilizing disulfide bridges or N-terminal thermostabilizing domain [14] , however, the aromatic residues in xylanase were found to be responsible for its thermal stability. When additional aromatic interaction was introduced into Family 11 xylanase, the thermostability of the enzyme was improved [15,16] . Furthermore, the N-terminal fusion peptide (51 amino acids) may also play a role in stabilizing its righthand ␤ -sandwich structure.…”