Fungal rutinosidases are retaining diglycosidases that release rutinose from flavonoid glycosides, catalyzing valuable transglycosylation reactions. We systematically compare the substrate specificity and synthetic capabilities of two structurally characterized fungal rutinosidases from Aspergillus niger ( An Rut) and A. oryzae ( Ao Rut), across a panel of natural, modified, and non‐natural glycosides. An Rut displays remarkable glycone promiscuity, efficiently hydrolyzing not only rutinosides and β‐glucopyranosides but also β‐galactopyranosides, β‐xylopyranosides, and α‐L‐arabinopyranosides. An Rut also processes diglycosides, such as rutinosides and isomaltosides. It catalyzes transglycosylations with non‐natural glycosides as donors, glycosylating alcoholic and phenolic acceptors, including transfer of β‐D‐galacto‐, β‐D‐xylo‐, and α‐L‐arabinopyranosyl residues. In contrast, Ao Rut exhibits a narrower substrate range: it recognizes rutinosides and selected monoglycosides as substrates but does not hydrolyze most other diglycosides. Unexpectedly, Ao Rut catalyzes efficient transglycosylation using p‐ nitrophenyl β‐D‐galactopyranoside and p NP‐α‐L‐arabinopyranoside as donors, producing novel β‐galacto‐ and α‐L‐arabinopyranosides of various phenolic acceptors. Both enzymes also catalyze donor self‐condensation products, such as p NP‐β‐D‐glucopyranosyl‐(1→3)‐β‐D‐glucopyranoside and p NP‐β‐D‐xylopyranosyl‐(1→3)‐β‐D‐xylopyranoside and p NP‐β‐D‐xylopyranosyl‐(1→2)‐β‐D‐xylopyranoside (only Ao Rut). Notably, this is the first reported case of a rutinosidase glycosylating a sugar acceptor. These findings reveal distinct glycone preferences and mechanistic features of the two enzymes and significantly expand the repertoire of glycosides accessible through fungal rutinosidases, highlighting their utility for synthesizing novel carbohydrate derivatives.
Brouzdová et al. (Tue,) studied this question.