[P002] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P01-124] Structural Determinants of the Exo-Mode of Action of BcXyn26A, a Novel β-1,3-Xylobiohydrolase Encoded in a Human Gut Bacterial Polysaccharide Utilization Locus for Macroalgal β-1,3-Xylan

○Kotone Yamamoto¹, Sanae Hori¹, Fumiyoshi Okazaki¹ (1. Department of Life Sciences, Graduate School of Bioresources, Mie University (Japan))

Keywords:

β-1,3-xylobiohydrolase,β-1,3-xylan,β-1,3-xylooligosaccharides,Bacteroides cellulosilyticus,human gut bacteria,polysaccharide utilization locus,macroalgae,prebiotics

β-1,3-Xylanase (1,3-β-D-xylan xylanohydrolase; EC 3.2.1.32) is an endo-type enzyme that randomly hydrolyzes β-1,3-xylan, a polysaccharide found exclusively in the cell walls of red and green algae. Although such enzymes have mainly been reported in marine bacteria, we identified two β-1,3-xylanase genes, xyn26A and xyn26B, located together within a putative β-1,3-xylan-specific polysaccharide utilization locus (PUL) in the human gut bacterium Bacteroides cellulosilyticus. The genes encoding BcXyn26A and BcXyn26B were codon-optimized, synthesized, and heterologously expressed in Escherichia coli to obtain recombinant enzymes. Both enzymes exhibited substrate specificity toward β-1,3-xylan. BcXyn26B exhibited endo-type activity, producing β-1,3-xylooligosaccharides with varying degrees of polymerization and showing higher activity than previously reported enzymes. In contrast, BcXyn26A exhibited exo-type activity, releasing only β-1,3-xylobiose. These results suggest that BcXyn26A is a novel exo-β-1,3-xylanase that can be classified as a β-1,3-xylobiohydrolase (1,3-β-D-xylan xylobiohydrolase; EC 3.2.1.-). Furthermore, the combined action of the two enzymes increased β-1,3-xylobiose production, demonstrating their cooperative function in the efficient degradation of β-1,3-xylan. Based on these findings and the genetic context of the PUL, the resulting β-1,3-xylobiose is likely transported into the cell and subsequently hydrolyzed into xylose by a putative β-1,3-xylosidase encoded within the same locus. Despite their high sequence similarity, the two enzymes exhibit distinct degradation modes. To elucidate the structural determinants underlying the exo-mode of action of BcXyn26A, we performed comparative structural analysis based on predicted three-dimensional models and mutational analysis. The results revealed that a characteristic β-turn near the minus subsites of the substrate-binding cleft, together with the E120 residue, plays a critical role in defining the exo-type activity of BcXyn26A. These findings highlight the functional and biotechnological significance of β-1,3-xylan and its hydrolysis products. β-1,3-Xylan may selectively promote the growth of beneficial Bacteroides species in the human gut, indicating its potential as a novel prebiotic. In addition, β-1,3-xylobiose, the minimal unit of β-1,3-linked xylosyl residues, exhibits high solubility and ease of handling. Our findings provide a basis for the development of microbial production systems for β-1,3-xylobiose and for the sustainable utilization of algal biomass in prebiotic and biotechnological applications.