Substrate specificity of Aspergillus oryzae family 3 β-glucosidase

Among glycoside hydrolases, β-glucosidase plays a unique role in many physiological and biocatalytical processes that involve the β-linked O-glycosyl bond of various oligomeric saccharides or glycosides. Structurally, the enzyme can be grouped into glycoside hydrolase family 1 and 3. Although the basic (“retaining, double-displacement”) mechanism for the catalysis of family 3 β-glucosidase has been established, in-depth understanding of its structure–function relationship, particularly the substrate specificity that is of great interest for developing the enzyme as a versatile biocatalyst, remains limited. To further probe the active site, we carried out a comparative study on a family 3 β-glucosidase from Aspergillus oryzae with substrates and competitive inhibitors of different structures, in attempt to evaluate the site-specific spatial and chemical interactions between a pyranosyl substrate and the enzyme. Our results showed the enzyme having a strict stereochemical requirement (to accommodate β-d-glucopyranose) for its “− 1” active subsite, in contrast to its family 1 counterpart.