Biochimica et Biophysica Acta. Proteins and Proteomics issue:1804 pages:977-985
Glycoside hydrolase family (GH) 11 xylanase A from Bacillus subtilis (BsXynA) was subjected to site-directed mutagenesis to probe the role of aglycon active site residues with regard to activity, binding of decorated substrates and hydrolysis product profile. Based on 3D structures of BsXynA in complex with substrate, active site amino acid residues interacting with the substrate were identified and modified. Several aromatic residues in the aglycon subsites that make strong substrate-protein interactions and are indispensable for enzyme activity, were also important for the specificity of the xylanase. In the +2 subsite of BsXynA, Tyr65 and Trp129 were identified as residues that govern the binding of decorated substrates. Most interestingly, replacement of Tyr88 by Ala in the +3 subsite created an enzyme able to produce a wider variety of hydrolysis products than wild type BsXynA. The contribution of the +3 subsite to the substrate specificity of BsXynA was established more in detail by mapping the enzyme binding site of the wild type xylanase and mutant Y88A with labelled xylo-oligosaccharides. Also, the length of the cord - a long loop flanking the aglycon subsites of GH11 xylanases - proved to impact the hydrolytic action of BsXynA. The aglycon side of the active site cleft of BsXynA, therefore, offers great potential for engineering and design of xylanases with a desired specificity.