Mutational analysis of the effects of N-glycosylation sites on the activity and thermal stability of rutinosidase from Aspergillus oryzae.

Purified recombinant rutinosidase from Aspergillus oryzae expressed in Pichia pastoris (rAoRutM) exhibits increase in thermal stability after treatment with endo-ß-N-acetylglucosaminidase H (endo-H). In this study, the role of N-glycosylation in the activity and thermal stability of rAoRutM was analyzed via site-directed mutagenesis. Based on the crystal structure of AoRutM, five N-glycosylation sites (N32, N128, N176, N288, and N359) were identified in the AoRut protein. Among five single variants constructed for these sites, the N128D, N176D, and N359D variants exhibited similar mobility bands compared to that of the wild-type enzyme based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas the N32D and N288D variants exhibited slightly and considerably increasing mobility bands, respectively. The N128D and N288D variants showed increasing and decreasing rutinosidase activity, respectively, compared to the case for the wild-type, without and with endo-H treatments. While the N128D and N176D variants had lower Km values, the N288D and N359D variants had higher Km values, compared to the wild-type, without and with endo-H treatments. Surprisingly, the N32D and N176D variants exhibited considerably greater thermal stability than the wild-type, without or with the endo-H treatments, whereas the N128D and N359D variants exhibited drastically decreased thermal stability. Circular dichroism (CD) spectra of the N128D and N359D variants showed a similar CD profile to that of the wild-type treated with endo-H; however, the molar ellipticity values of the peaks at 208 nm and 212 nm in the above variants varied from those of the intact wild-type and other variants.

Aspergillus oryzae Rutinosidase: Biochemical and Structural Investigation.

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 (ß/α)8 TIM barrel fold with structural similarity to that of rutinosidase from Aspergillus niger (AnRut) and an exo-ß-(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-ß-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 ß-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.