Fungal Rutinosidase─Engineering of a Side Tunnel Steers Its Transglycosylation Potential

Rutinosidases (α-l-rhamnosyl-β-d-glucosidases) are glycosidases (EC 3.2.1.168) that cleave the glycosidic bond between the aglycone and the disaccharide residue rutinose. Their dual substrate specificity is reflected in their activity toward both rutin (rutinosylated) and isoquercitrin (glucosylated) substrates. The structure of rutinosidase from Aspergillus niger (AnRut) features a side tunnel that influences the enzyme hydrolytic and transglycosylation activities. We present a mutagenesis study of this side tunnel, and the compartment forming the +1 binding subsite, resulting in seven variants with different active-site entry geometries. We show that the key side tunnel residues affect the catalytic and trans-rutinosylation potential of AnRut and compare these properties with rutinosidase from Aspergillus oryzae, which has a side groove instead of a side tunnel. The trans-rutinosylation abilities of the enzymes were tested using a diverse library of acceptors. This work expands the structure-function understanding of fungal rutinosidases and underlies the hypothesis that the engineering of the side tunnel can increase the transglycosylation-to-hydrolysis ratio.