The rutinosidase (Rut)-encoding gene Aorut has been expressed in Pichia pastoris with its native signal sequence from Aspergillus oryzae Biochemical and structural investigation of the purified recombinant mature A. oryzae Rut (AoRut), designated rAoRutM, was performed in this study. A 1.7-Å resolution crystal structure of rAoRutM was determined, which is an essential step forward in the utilization of AoRut as a potential catalyst. The crystal structure of rAoRutM was represented by a (β/α)8 TIM barrel fold with structural similarity to that of rutinosidase from Aspergillus niger (AnRut) and an exo-β-(1,3)-glucanase from Candida albicans The crystal structure revealed that the catalytic site was located in a deep cleft, similarly to AnRut, and that internal cavities and water molecules were also present. Purified rAoRutM hydrolyzed not only 7-O-linked and 3-O-linked flavonoid rutinosides but also 7-O-linked and 3-O-linked flavonoid glucosides. rAoRutM displayed high catalytic activity toward quercetin 3-O-linked substrates such as rutin and isoquercitrin, rather than to the 7-O-linked substrate, quercetin-7-O-glucoside. Unexpectedly, purified rAoRutM exhibited increased thermostability after treatment with endo-β-N-acetylglucosaminidase H. Circular dichroism (CD) spectra of purified intact rAoRutM and of the enzyme after N-deglycosylation showed a typical α-helical CD profile; however, the molar ellipticity values of the peaks at 208 nm and 212 nm differed. The Km and kcat values for the substrates modified by rutinose were higher than those for the substrates modified by β-d-glucose.IMPORTANCE 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 A. oryzae rutinosidase provides a potential biocatalyst for industrial applications of flavonoids.
Keywords: Aspergillus oryzae; N-glycosylation; crystal structure; rutinosidase; substrate specificity.
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