title: Modular glucuronoxylan-specific xylanase with a family CBM35 carbohydrate-binding module.
abstract: Xyn30D from the xylanolytic strain Paenibacillus barcinonensis has been identified and characterized. The enzyme shows a modular structure comprising a catalytic module family 30 (GH30) and a carbohydrate-binding module family 35 (CBM35). Like GH30 xylanases, recombinant Xyn30D efficiently hydrolyzed glucuronoxylans and methyl-glucuronic acid branched xylooligosaccharides but showed no catalytic activity on arabinose-substituted xylans. Kinetic parameters of Xyn30D were determined on beechwood xylan, showing a K(m) of 14.72 mg/ml and a k(cat) value of 1,510 min(-1). The multidomain structure of Xyn30D clearly distinguishes it from the GH30 xylanases characterized to date, which are single-domain enzymes. The modules of the enzyme were individually expressed in a recombinant host and characterized. The isolated GH30 catalytic module showed specific activity, mode of action on xylan, and kinetic parameters that were similar to those of the full-length enzyme. Computer modeling of the three-dimensional structure of Xyn30D showed that the catalytic module is comprised of a common (β/α)(8) barrel linked to a side-associated β-structure. Several derivatives of the catalytic module with decreasing deletions of this associated structure were constructed. None of them showed catalytic activity, indicating the importance of the side β-structure in the catalysis of Xyn30D. Binding properties of the isolated carbohydrate-binding module were analyzed by affinity gel electrophoresis, which showed that the CBM35 of the enzyme binds to soluble glucuronoxylans and arabinoxylans. Analysis by isothermal titration calorimetry showed that CBM35 binds to glucuronic acid and requires calcium ions for binding. Occurrence of a CBM35 in a glucuronoxylan-specific xylanase is a differential trait of the enzyme characterized.
authors.affiliation: Department of Microbiology, University of Barcelona, Barcelona, Spain.
authors.forename: Susana Valeria
authors.initials: SV
authors.lastname: Valenzuela
authors.forename: Pilar
authors.initials: P
authors.lastname: Diaz
authors.forename: F I Javier
authors.initials: FI
authors.lastname: Pastor
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D056507:Paenibacillus / Q000201:enzymology* / Q000235:genetics; D011134:Polysaccharides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D014990:Xylans / Q000737:chemistry / Q000378:metabolism*; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D011134:Polysaccharides; D011994:Recombinant Proteins; D014990:Xylans; C038910:glucuronoxylan; D014995:Xylosidases