Abstract
A GH8 family enzyme involved in xylan depolymerization has been characterized. The enzyme, Rex8A, is a reducing-end xylose-releasing exo-oligoxylanase (Rex) that efficiently hydrolyzes xylooligosaccharides and shows minor activity on polymeric xylan. Rex8A hydrolyzes xylooligomers of 3 to 6 xylose units to xylose and xylobiose in long-term incubations. Kinetic constants of Rex8A were determined on xylotriose, showing a Km of 1.64 ± 0.03 mM and a kcat value of 118.8 s(-1) Besides linear xylooligosaccharides, the enzyme hydrolyzed decorated xylooligomers. The catalytic activity on branched xylooligosaccharides, i.e., the release of xylose from the reducing end, is a newly described trait of xylose-releasing exo-oligoxylanases, as the exo-activity on these substrates has not been reported for the few of these enzymes characterized to date. Modeling of the three-dimensional (3D) structure of Rex8A shows an (α/α)6 barrel fold where the loops connecting the α-helices contour the active site. These loops, which show high sequence diversity among GH8 enzymes, shape a catalytic cleft with a -2 subsite that can accommodate methyl-glucuronic acid decorations. The hydrolytic ability of Rex8A on branched oligomers can be crucial for the complete depolymerization of highly substituted xylans, which is indispensable to accomplish biomass deconstruction and to generate efficient catalysts. Importance: A GH8 family enzyme involved in xylan depolymerization has been characterized. The Rex8A enzyme from Paenibacillus barcinonensis is involved in depolymerization of glucuronoxylan, a major component of the lignocellulosic substrates. The study shows that Rex8A is a reducing-end xylose-releasing exo-oligoxylanase that efficiently hydrolyzes xylose from neutral and acidic xylooligosaccharides generated by the action of other xylanases also secreted by the strain. The activity of a Rex enzyme on branched xylooligosaccharides has not been described to date. This report provides original and useful information on the properties of a new example of the rarely studied Rex enzymes. Depolymerization of highly substituted xylans is crucial for biomass valorization as a platform for generation of biofuels, chemicals, and solvents.