Graduate School of Science and Engineering, Faculty of Engineering, Yamagata University, Yonezawa, Japan
Department of Biosciences, Faculty of Agriculture, Yamagata University, Tsuruoka, Japan.
Appl Environ Microbiol. 2021 Jan 15;87(3). doi: 10.1128/AEM.02438-20.
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.
本研究在 中表达了具有其天然信号序列的木二糖酶（Rut）编码基因。对纯化的重组成熟木二糖酶（Rut）（命名为 rRutM）进行了生化和结构研究。该研究确定了 rRutM 的 1.7Å 分辨率晶体结构，这是将 Rut 用作潜在催化剂的重要一步。rRutM 的晶体结构由（β/α）TIM 桶折叠表示，与来自 （Rut）的木二糖酶和来自 的外切-β-（1,3）-葡聚糖酶具有结构相似性。晶体结构表明，催化位点位于深裂缝中，类似于 Rut，并且存在内部空腔和水分子。纯化的 rRutM 不仅水解 7--连接和 3--连接的黄酮木二糖苷，还水解 7--连接和 3--连接的黄酮葡萄糖苷。rRutM 对 quercetin 3--连接的底物（如芦丁和异槲皮苷）具有高催化活性，而对 7--连接的底物 quercetin-7--glucoside 则没有。出乎意料的是，经内切-β--乙酰氨基葡萄糖苷酶 H 处理后，纯化的 rRutM 表现出增强的热稳定性。纯化的完整 rRutM 和酶经 -去糖基化后的圆二色（CD）光谱显示出典型的α-螺旋 CD 谱；然而，在 208nm 和 212nm 处的峰的摩尔椭圆度值不同。rutinose 修饰的底物的 和 值高于β-d-葡萄糖修饰的底物的 值。类黄酮糖苷是广泛存在于自然界中的一类次生代谢产物。这些化合物分别参与植物性食品或饮料的苦味或混浊。类黄酮糖苷的降解可以通过两种替代的酶促途径进行：一种由单糖苷酶介导，另一种由二糖苷酶催化。本报告对 木二糖酶的生化和结构研究为类黄酮的工业应用提供了一种潜在的生物催化剂。
