Cryo‐ <scp>EM</scp> structure of <scp>GH43</scp> β‐Xylosidase from <i>Enterobacter cloacae</i> provides insights into substrate specificity and the role of an auxiliary domain in enzymatic activity

Xylan is the second most abundant polysaccharide in plant cell walls, consisting of a β-1,4-linked xylopyranosyl backbone that can be substituted with various side chains. Depolymerization of xylan is predominantly catalyzed by the coordinated activity of β-xylanases and β-xylosidases. In this study, we determined the cryo-electron microscopy (cryo-EM) structure of Enterobacter cloacae β-xylosidase (EcXyl43), a glycoside hydrolase family 43 (GH43) enzyme. Additionally, we resolved the X-ray crystal structure of a catalytically inactive F507A mutant of EcXyl43. Together, these structures represent the first structural characterization of a β-xylosidase from the Enterobacter genus using both X-ray diffraction and cryoEM. Furthermore, to elucidate the molecular basis of substrate recognition and specificity, we conducted molecular dynamics simulations of the enzyme. Structural and computational analysis of EcXyl43 identified key determinants of the enzyme's preference for longer xylooligosaccharides and revealed how noncatalytic residues within the auxiliary domain modulate its activity.