Aspergillus oryzae Rutinosidase: Biochemical and Structural Investigation.

The rutinosidase (Rut)-encoding gene has been expressed in with its native signal sequence from Biochemical and structural investigation of the purified recombinant mature Rut (Rut), designated rRutM, was performed in this study. A 1.7-Å resolution crystal structure of rRutM was determined, which is an essential step forward in the utilization of Rut as a potential catalyst. The crystal structure of rRutM was represented by a (β/α) TIM barrel fold with structural similarity to that of rutinosidase from (Rut) and an exo-β-(1,3)-glucanase from The crystal structure revealed that the catalytic site was located in a deep cleft, similarly to Rut, and that internal cavities and water molecules were also present. Purified rRutM hydrolyzed not only 7--linked and 3--linked flavonoid rutinosides but also 7--linked and 3--linked flavonoid glucosides. rRutM displayed high catalytic activity toward quercetin 3--linked substrates such as rutin and isoquercitrin, rather than to the 7--linked substrate, quercetin-7--glucoside. Unexpectedly, purified rRutM exhibited increased thermostability after treatment with endo-β--acetylglucosaminidase H. Circular dichroism (CD) spectra of purified intact rRutM and of the enzyme after -deglycosylation showed a typical α-helical CD profile; however, the molar ellipticity values of the peaks at 208 nm and 212 nm differed. The and values for the substrates modified by rutinose were higher than those for the substrates modified by β-d-glucose. Flavonoid glycosides constitute a class of secondary metabolites widely distributed in nature. These compounds are involved in bitter taste or clouding in plant-based foods or beverages, respectively. Flavonoid glycoside degradation can proceed through two alternative enzymatic pathways: one that is mediated by monoglycosidases and another that is catalyzed by a diglycosidase. The present report on the biochemical and structural investigation of rutinosidase provides a potential biocatalyst for industrial applications of flavonoids.