Novel molecular strategies for xylan degradation learned from Xanthomonas phytopathogens

The xylan CUT system, conserved among Xanthomonas ssp., contains two major xylanase-related genes, XynA and XynB, which are important for nutrient uptake and bacterial adaptation to the phyllosphere. However, the molecular bases for their action on plant cell-wall polysaccharides are still elusive. Herein, we demonstrated that XynA is the first dimeric GH10 member with reducing end xylose-releasing exo-oligoxylanase activity, being inactive against polymeric xylans. Structural analysis revealed that an insertion in the β7-α7 loop promotes a physical barrier at the +2 subsite conferring its unique action mode within the GH10 family. A single mutation that impaired dimerization became the enzyme active against xylan chains and this effect was 20-fold higher when this loop was tailored to match a canonical sequence of endo-β-1,4-xylanases, supporting our mechanistic model. On the other hand, the most divergent xylanase-related protein, XynB, showed a typical endo-β-1,4-xylanase activity with a fully conserved glycone-binding region and a unique product release area. This enzyme contains a calcium ion bound to a motif located between the β2-α2 loop and the α3 helix that is not conserved in other GH10 homologues. This ion is required for full catalytic activity and confers remarkable structural stability to XynB (ΔT_M~ 11ºC). These findings reveal new molecular strategies for hemicellulose deconstruction expanding our knowledge regarding the activities and regulatory mechanisms found in the GH10 family and highlighting the great potential of plant bacterial pathogens as a source of biotechnologically-relevant enzymes.