Coevolution of carbohydrate-binding and catalytic modules revealed by a novel distant subgroup of CBM22 in a highly...

GH10 endoxylanases exhibit significant structural and modular diversity, with many members containing carbohydrate-binding modules (CBMs) whose functions remain to be elucidated. In this study, we identified a GH10 endoxylanase PphXyn10B from the xylanolytic bacterium Paenibacillus physcomitrellae XB. Predictions by AlphaFold3 and modeling by SWISS-MODEL suggested that its N-terminus contains two novel CBM22 modules (nCBM22s). To investigate the functions of this enzyme and its nCBM22s, we heterologously expressed the full-length recombinant enzyme (rnCBM22s-Xyn10B), truncated variants containing only a single nCBM22, both two nCBM22s (rnCBM22s), and retaining only the intact GH10 catalytic domain (CD, rXyn10B). The full-length recombinant efficiently hydrolyzed beechwood xylan (BWX) and arabinoxylan (AX), with xylobiose as the main oligosaccharide end product. Deletion of the two nCBM22s did not significantly alter the product profile or hydrolysis characteristics, except for an increase in the initial reaction rate. Notably, the two nCBM22s exhibited broad carbohydrate-binding capacity, recognizing not only insoluble BWX but also soluble AX and glucuronoxylan. Combined phylogenetic and domain analyses revealed that CBM22s in GH10 xylanases from different strains are highly diverse, with CBM22s from different subgroups being linked to the corresponding subgroups of GH10 CDs. The majority of nCBM22s clustered into a branch and were exclusively associated with the GH10_e130 CDs, suggesting that they may represent an evolutionary type among the various annotated CBM22 subgroups. This study is of great significance for understanding the diversity of the CBM22 family and its co-evolution with GH10 CDs, as well as for deepening our knowledge of the action mechanisms of efficient hydrolases.

GH10 endoxylanases are ubiquitously distributed across microbial taxa and play essential roles in depolymerizing structurally heterogeneous xylans in plant cell walls. The discovery and systematic characterization of GH10 enzymes with potent activity against recalcitrant xylans are critical for advancing their application in lignocellulosic biomass conversion and sustainable bioenergy production. In this study, a GH10 endoxylanase, PphXyn10B, from Paenibacillus physcomitrellae strain XB, exhibited broad substrate specificity and high catalytic efficiency toward various xylans, including beechwood xylan, wheat arabinoxylan, and xylooligosaccharides, yielding xylobiose as the predominant hydrolysis product. Furthermore, this enzyme contains two novel CBM22 modules and forms a phylogenetically distinct and well-supported lineage within the GH10_e130 catalytic domain (CD), evolutionarily separate from the known CBM22-associated GH10 CD. Collectively, these findings enhance our understanding of modular coevolution in GH10 xylanases and provide a mechanistic foundation for the rational engineering of multi-domain xylanolytic biocatalysts.