Modular Glucuronoxylan-Specific Xylanase with a Family CBM35 Carbohydrate-Binding Module

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Xyn30D from the xylanolytic strain<jats:named-content content-type="genus-species">Paenibacillus barcinonensis</jats:named-content>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<jats:italic>K<jats:sub>m</jats:sub></jats:italic>of 14.72 mg/ml and a<jats:italic>k</jats:italic><jats:sub>cat</jats:sub>value of 1,510 min<jats:sup>−1</jats:sup>. 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)<jats:sub>8</jats:sub>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.</jats:p>

The Glycoside Hydrolase Family 8 Reducing-End Xylose-Releasing Exo-oligoxylanase Rex8A from Paenibacillus barcinonensis BP-23 Is Active on Branched Xylooligosaccharides

“…A DNA fragment with sequence homology to GH8 enzyme-encoding genes was identified upstream of xyn11E of Paenibacillus barcinonensis BP-23 (CECT 7022; DSM 15478). Its complete sequence was obtained using genome walking with this DNA fragment and a GenomeWalker universal kit (Clontech) as described previously (10). To amplify rex8A, primers Rex8A-fw (5=-AGA GGAGGAGCCCATGGACATAACAGGAAAAGGC-3=) and Rex8A-bw (5=-AAAAAAAAGCTTATACACTCGGTAGTTACCA-3=) (restriction sites are underlined) were used (Kapa-HiFi; KAPABiosystems).…”

“…Most xylanases are highly active on unsubstituted regions of xylan but have diminished activity on decorated xylans (6,7). However, a few examples of xylanases, all belonging to the GH30 family, have been shown to be dependent on methyl-glucuronic acid substitutions for activity, being considered glucuronoxylanases (8)(9)(10). A different decoration requirement has been reported for the GH5 xylanase Xyl5A from Clostridium thermocellum that shows specificity for arabinose-substituted xylan (11).…”

Structural Analysis of Glucuronoxylan-specific Xyn30D and Its Attached CBM35 Domain Gives Insights into the Role of Modularity in Specificity*

“…Cloning, Expression, and Purification-Construction of Xyn30D, Xyn30D-GH30, and Xyn30D-CBM35 expression vectors were previously described (27). The protein samples were purified from Escherichia coli BL21Star (DE3) recombinant cultures containing plasmids pET101Xyn30D, pET101Xyn30D-GH30, and pET28aXyn30D-CBM35, respectively.…”

“…Like the few examples of characterized glucuronoxylanases of family GH30, the enzyme shows requirement of methylglucuronic substitutions for catalysis and is not active on arabinoxylans (27). However, unlike the structurally characterized GH30 glucuronoxylanases from Bacillus subtilis and Erwinia chrysanthemi (28,29), which are single domain enzymes, Xyn30D is a modular enzyme.…”

Mannan specific family 35 carbohydrate-binding module (CtCBM35) of Clostridium thermocellum: structure analysis and ligand binding

“…Ghosh et al tein structures (Boraston et al 2004). In general, most family CBM35 acquired a type B conformation in nature (Valenzuela et al 2012). Type B modules act more in diverse fashion of ligand specificity than the other types of CBMs, which showed wide range of affinities toward cellulose, xylan, β-(1,3)-glucans, β-(1,3)-(1,4) mixed-linkage glucans, starch, glucomannan and mannan (Tunnicliffe et al 2005).…”