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 K_m and k_cat values for the substrates modified by rutinose were higher than those for the substrates modified by β-d-glucose.

中文翻译：

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米曲霉芸香糖苷酶：生化和结构研究

芦丁糖苷酶（Rut）编码基因Aorut已在巴斯德毕赤酵母中表达，其天然信号序列来自米曲霉。在这项研究中进行了纯化的重组成熟米曲霉Rut（Ao Rut）的生化和结构研究，命名为r Ao RutM。确定了r Ao RutM的1.7Å分辨率晶体结构，这是利用Ao Rut作为潜在催化剂迈出的重要一步。r Ao RutM的晶体结构由（β/α）₈ TIM桶形折叠表示，其结构与来自黑曲霉（一种车辙）和外切β-（1,3） -葡聚糖酶由白色念珠菌。晶体结构表明，催化部位位于深裂缝中，与An Rut相似，并且还存在内部空腔和水分子。纯化[R敖RutM水解不仅7 Ø联并且3- Ø -连接的类黄酮芸香苷，而且7 Ø联并且3- Ø -连接黄酮苷。r Ao RutM对槲皮素3- O连接的底物（如芦丁和异槲皮苷）显示出高催化活性，而不是7- O-连接的底物，槲皮素-7- O-葡萄糖苷。出乎意料的是，纯化的[R敖RutM表现出与Endo-β-治疗后增加的热稳定性Ñ -acetylglucosaminidase纯化完整的r H.圆二色性（CD）谱鳌RutM和酶后Ñ -deglycosylation呈典型的α螺旋CD轮廓; 但是，在208nm和212nm处的峰的摩尔椭圆率值不同。芦丁糖修饰的底物的K _m和k _cat值高于β- d-葡萄糖修饰的底物的K _m和k _cat值。