Mode of action of glycoside hydrolase family 5 glucuronoxylan xylanohydrolase from Erwinia chrysanthemi

Abstract

The mode of action of xylanase A from a phytopathogenic bacterium, Erwinia chrysanthemi, classified in glycoside hydrolase family 5, was investigated on xylooligosaccharides and polysaccharides using TLC, MALDI‐TOF MS and enzyme treatment with exoglycosidases. The hydrolytic action of xylanase A was found to be absolutely dependent on the presence of 4‐O‐methyl‐d‐glucuronosyl (MeGlcA) side residues in both oligosaccharides and polysaccharides. Neutral linear β‐1,4‐xylooligosaccharides and esterified aldouronic acids were resistant towards enzymatic action. Aldouronic acids of the structure MeGlcA3Xyl3 (aldotetraouronic acid), MeGlcA3Xyl4 (aldopentaouronic acid) and MeGlcA3Xyl5 (aldohexaouronic acid) were cleaved with the enzyme to give xylose from the reducing end and products shorter by one xylopyranosyl residue: MeGlcA2Xyl2, MeGlcA2Xyl3 and MeGlcA2Xyl4. As a rule, the enzyme attacked the second glycosidic linkage following the MeGlcA branch towards the reducing end. Depending on the distribution of MeGlcA residues on the glucuronoxylan main chain, the enzyme generated series of shorter and longer aldouronic acids of backbone polymerization degree 3–14, in which the MeGlcA is linked exclusively to the second xylopyranosyl residue from the reducing end. Upon incubation with β‐xylosidase, all acidic hydrolysis products of acidic oligosaccharides and hardwood glucuronoxylans were converted to aldotriouronic acid, MeGlcA2Xyl2. In agreement with this mode of action, xylose and unsubstituted oligosaccharides were essentially absent in the hydrolysates. The E. chrysanthemi xylanase A thus appears to be an excellent biocatalyst for the production of large acidic oligosaccharides from glucuronoxylans as well as an invaluable tool for determination of the distribution of MeGlcA residues along the main chain of this major plant hemicellulose.

Consolidation of glycosyl hydrolase family 30: A dual domain 4/7 hydrolase family consisting of two structurally distinct groups

“…With respect to the well-characterized, GH family 5 endoxylanase [4,5,11,13], several authors have previously noted this classification anomaly [7,[9][10][11]. The findings presented in this work support a reclassification of these GH family 5 enzyme groups as GH30 enzymes.…”

“…A recent report [41] assigned an acidic b-1,4-xylanase function to a fungal enzyme that shares approximately 50% identity with a subgroup G enzyme, also of fungal origin (ACC B8M989). No information is available for the sparsely populated subgroup F. Two subgroup H members are characterized having glucuronoxylan xylanohydrolase activity and structure models are available for these same enzymes [4][5][6]11,42].…”

“…In contrast, subgroups G and H both have assigned xylanase activity. However, subgroup H xylanases are fundamentally unique in comparison to subgroup G having an enzymatic activity which relies upon recognition of an a-1,2-linked 4-O-methyl glucuronate moiety [4,5,41].…”

“…The Carbohydrate-Active EnZymes database (CAZy) (http://www.cazy.org) [1][2][3] is the primary resource for sequence based categorization of enzymes that catalyze reactions involving the cleavage, creation or modification of glycosidic bonds. Recent biochemical characterization [4,5] and structural studies of the GH5 xylanase XynC from Bacillus subtilis [6] has directed attention to a previously noted inconsistency in the GH classification of this enzyme [7][8][9][10][11]. Early classification discussed the difficulty of making an unambiguous assignment based on hydrophobic cluster analysis comparisons with the XynC homolog, XynA from Erwinia chrysanthemi with GH family 5 and 30 (b/a) 8 barrel domains [12,13].…”

The Biochemistry and Structural Biology of Plant Cell Wall Deconstruction

“…By contrast, an arabinoxylan-specific GH43 arabinofuranosidase, which removes O2-or O3-linked Araf side chains from singularly substituted Xyl residues, contains a small substrate-binding pocket embedded in a shallow cleft that is optimized to bind the 3-fold helical structure of the xylan backbone (Vandermarliere et al, 2009). Recent protein crystallographic studies have shown that xylan side chains can be accommodated and can actually be exploited as specificity determinants (Pell et al, 2004;Vardakou et al, 2005), while a GH5 xylanase displays an absolute requirement for 4-O-methyl-D-GlcUA appended to the Xyl positioned at the 22 subsite (Vrsanska et al, 2007). There are two structures of this enzyme (Larson et al, 2003;St John et al, 2009); however, the mechanism by which the enzyme recognizes the uronic acid side chain remains unclear.…”