title: The role of the glucuronoxylan carboxyl groups in the action of endoxylanases of three glycoside hydrolase families: A study with two substrate mutants.
abstract: BACKGROUND
  Bacterial 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.
  
  
  METHODS
  In 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.
  
  
  RESULTS
  Elimination 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.
  
  
  CONCLUSIONS
  These 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.
  
  
  CONCLUSIONS
  The uncharged glucuronoxylan derivatives will be useful to differentiate GH30 xylanases with various degree of selectivity for glucuronoxylan, including fungal enzymes without the conserved arginine.
authors.affiliation: Institute of Chemistry, Slovak Academy of Sciences, Bagvaerd, Denmark. Electronic address: chempbsa@savba.sk.
authors.forename: P
authors.initials: P
authors.lastname: Biely
authors.affiliation: Institute of Chemistry, Slovak Academy of Sciences, Bagvaerd, Denmark.
authors.forename: A
authors.initials: A
authors.lastname: Malovíková
authors.affiliation: Institute of Chemistry, Slovak Academy of Sciences, Bagvaerd, Denmark.
authors.forename: J
authors.initials: J
authors.lastname: Hirsch
authors.affiliation: Novozymes A/S, Bagvaerd, Denmark.
authors.forename: K B R
authors.initials: KB
authors.lastname: Morkeberg Krogh
authors.affiliation: Institute of Chemistry, Slovak Academy of Sciences, Bagvaerd, Denmark.
authors.forename: A
authors.initials: A
authors.lastname: Ebringerová
mesh_terms: D043364:Endo-1,4-beta Xylanases / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014990:Xylans / Q000737:chemistry*
chemical_list: D014990:Xylans; C038910:glucuronoxylan; D006026:Glycoside Hydrolases; D043364:Endo-1,4-beta Xylanases
keywords: 4-O-methylglucoxylan; 4-O-methylglucuronic acid; Carboxyl group; Endoxylanase; Glucuronoxylan; Glycoside hydrolase families; Mode of action