The role of the glucuronoxylan carboxyl groups in the action of endoxylanases of three glycoside hydrolase families: A study with two substrate mutants

• Action of endoxylanases (EXs) was examined on novel glucuronoxylan derivatives.
• One was its methyl ester and the second reduced ester, 4-O-methyl-d-glucoxylan.
• Elimination of the free carboxyl group did not affect significantly GH10 and GH11 EXs.
• Both modifications resulted in deep drop of specific activity of a bacterial GH30 EX.
• Interaction of the GH30 EX with the uronic acid carboxyl is crucial for its activity.

BackgroundBacterial appendage-dependent GH30 glucuronoxylan hydrolases recognize the substrate through an ionic interaction of a conserved positively charged arginine with the carboxyl group of 4-O-methyl-d-glucuronic acid. One of the options to verify this interaction is preparation of enzyme mutants. An alternative approach is a chemical modification of the substrate, glucuronoxylan, in which the free carboxyl group in all residues of MeGlcA is eliminated.MethodsIn this work the carboxyl groups of 4-O-methyl-d-glucuronic acid residues of an alkali extracted beechwood xylan were esterified with methanol. A water-soluble fraction of the polysaccharide methyl ester was converted by NaBH4 reduction to the second soluble derivative, 4-O-methylglucoxylan. Specific activities of several endoxylanases (EXs) of GH families 10, 11 and 30 were determined on glucuronoxylan, and its two new uncharged derivatives.ResultsElimination of the free carboxyl group from the polysaccharide did not influence activities of GH10 EXs, but resulted in 50% decrease of specific activity of GH11 EXs, and led to more than 300-fold reduction of specific activity of Erwinia chrysanthemi GH30 xylanase.ConclusionsThese results confirm the crucial role of the interactions between GH30 xylanases and the MeGlcA carboxyl group for efficient cleavage of the polysaccharide. Analysis of the hydrolysis products by TLC and MS confirmed that all three types of xylanases hydrolyzed uncharged glucuronoxylans similarly as the original one.SignificanceThe uncharged glucuronoxylan derivatives will be useful to differentiate GH30 xylanases with various degree of selectivity for glucuronoxylan, including fungal enzymes without the conserved arginine.

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