Structural and biochemical characterization of a GH5_22 enzyme from the seaweed-derived thermophile Geobacillus thermodenitrificans OS27

Abstract

Geobacillus thermodenitrificans OS27 is a seaweed-derived thermophile that harbors a GH5_22 gene (bxlA) that encodes for a glycoside hydrolase fused with the cyclin box domain. In this study, we characterized the catalytic activity, enzymatic properties, three-dimensional structure, and physiological role of the gene product, GtBxlA. The enzyme was produced as a thermostable dimer, acting on p-nitrophenyl-β-d-xylopyranoside among 23 substrates. β-1,4-Linked xylooligosaccharides were also hydrolyzed from the nonreducing end. The activity indicated that GtBxlA functions as an exo-β-1,4-xylosidase. We determined the crystal structure of the Glu188Ala variant complexed with β-1,4-xylotriose at 1.52 Å resolution. GtBxlA exhibited an atypical (β/α)₈-barrel architecture. The cyclin box constituted a single α-helix within the core barrel and a lid-like domain that contributes to dimer formation. Structural analysis revealed that Glu188 and Glu318 are positioned to serve as the acid/base and nucleophile catalysts, respectively. Alanine mutagenesis confirmed the essential role of Glu188 and Glu318 in catalysis. We also determined the ligand-free structure of the Glu188Ala variant. Both structures were almost identical; however, a loop at the substrate entry site was fixed in the ligand-free structure, suggesting a conformational change upon substrate binding. Although bxlA deletion did not affect β-1,4-xylan utilization, its expression was induced by β-1,4-xylan. These observations suggest that G. thermodenitrificans OS27 employed GtBxlA in utilizing β-1,4-xylan. Notably, GtBxlA could hydrolyze β-1,3-linked xylooligosaccharides. This highlights the possibility that GtBxlA also assists the host in utilizing β-1,3-xylan, which is abundant in certain seaweeds.