Tryptic Mapping Based Structural Insights of Endo-1, 4-ß-Xylanase from Thermomyces lanuginosus VAPS-24.

An endo-1,4-ß-xylanase, XynA, from Thermomyces lanuginosus VAPS-24, was purified to homogeneity and exhibited a molecular mass of approximately 20 kDa. The protein sequence of XynA was found to be similar to those of other Thermomyces lanuginosus derived xylanases and, as a result, could be used as a model enzyme for understanding the protein structure-activity relationship and facilitating protein engineering to design enzyme variants with desirable properties. Therefore, this xylanase will be an attractive candidate for applications in the biofuel and fine chemical industries for the degradation of xylans in steam pre-treated biomass.

Endo-1,4-ß-xylanase-containing glycoside hydrolase families: characteristics, singularities and similarities.

Endo-1,4-ß-xylanases (EC 3.2.1.8) are O-glycoside hydrolases that cleave the internal ß-1,4-D-xylosidic linkages of the complex plant polysaccharide xylan. They are produced by a vast array of organisms where they play critical roles in xylan saccharification and plant cell wall hydrolysis. They are also important industrial biocatalysts with widespread application. A large and ever growing number of xylanases with wildly different properties and functionalites are known and a better understanding of these would enable a more effective use in various applications. The Carbohydrate-Active enZYmes database (CAZy), which classifies evolutionarily related proteins into a glycoside hydrolase family-subfamily organisational scheme has proven powerful in understanding these enzymes. Nevertheless, ambiguity currently exists as to the number of glycoside hydrolase families and subfamilies harbouring catalytic domains with true endoxylanase activity and as to the specific characteristics of each of these families/subfamilies. This review seeks to clarify this, identifying 9 glycoside hydrolase families containing enzymes with endo-1,4-ß-xylanase activity and discussing their properties, similarities, differences and biotechnological perspectives. In particular, substrate specificities and hydrolysis patterns and the structural determinants of these are detailed, with taxonomic aspects of source organisms being also presented. Shortcomings in current knowledge and research areas that require further clarification are highlighted and suggestions for future directions provided. This review seeks to motivate further research on these enzymes and especially of the lesser known endo-1,4-ß-xylanase containing families. A better understanding of these enzymes will serve as a foundation for the knowledge-based development of process-fitted endo-1,4-ß-xylanases and will accelerate their development for use with even the most recalcitrant of substrates in the biobased industries of the future.

Structural analysis of the reducing-end xylose-releasing exo-oligoxylanase Rex8A from Paenibacillus barcinonensis BP-23 deciphers its molecular specificity.

Reducing-end xylose-releasing exo-oligoxylanases (Rex) are GH8 enzymes that depolymerize xylooligosaccharides complementing xylan degradation by endoxylanases in an exo manner. We have studied Paenibacillus barcinonensis Rex8A and showed the release of xylose from xylooligomers decorated with methylglucuronic acid (UXOS) or with arabinose (AXOS). This gives the enzyme a distinctive trait among known Rex, which show activity only on linear xylooligosaccharides. The structure of the enzyme has been solved by X-ray crystallography showing a (α/α)6 folding common to GH8 enzymes. Analysis of inactived Rex8A-E70A complexed with xylotetraose revealed the existence of at least four binding subsites in Rex8A, with the oligosaccharide occupying subsites -3 to +1. The enzyme shows an extended Leu320-His321-Pro322 loop, common to other Rex, which blocks the binding of longer substrates to positive subsites further than +1 and seems responsible for the lack or diminished activity of Rex enzymes on xylan. Mutants with smaller residues in this loop failed to increase Rex8A activity on the polymer. Analysis of the complexes with AXOS showed the accommodation of arabinose at subsite -2, which cannot be allocated at subsite -1. Arabinose substitutions at the xylose O2 or O3 are accommodated by hydrophobic interaction and seem tolerated rather than recognized by Rex8A. A strained binding of the branch is facilitated by the lack of direct polar interactions of the xylose occupying this subsite, its water-mediated links allowing some conformational flexibility of the sugar. The plasticity of Rex8A is a notable property of the enzyme for its application in xylan deconstruction and upgrading. DATABASE: Structural data are available in PDB database under the accession numbers 6SRD (native form), 6TPP (E70A mutant in complex with EDO), 6TOW (E70A in complex with Xyl4), 6SUD (L320A mutant in complex with xylose), 6SHY (L320A/H321S double mutant in complex with EDO), 6TO0 (E70A in complex with AX3), and 6TRH (E70A in complex with AX4).