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Rutinosidase from Aspergillus niger: crystal structure and insight into the enzymatic activity.
The FEBS Journal ( IF 5.4 ) Pub Date : 2020-01-13 , DOI: 10.1111/febs.15208
Petr Pachl 1 , Jana Kapešová 2 , Jiří Brynda 1, 3 , Lada Biedermannová 4 , Helena Pelantová 2 , Pavla Bojarová 2 , Vladimír Křen 2 , Pavlína Řezáčová 1, 3 , Michael Kotik 2
Affiliation  

Rutinosidases (α‐l ‐rhamnosyl‐β‐d ‐glucosidases) catalyze the cleavage of the glycosidic bond between the aglycone and the disaccharide rutinose (α‐l ‐rhamnopyranosyl‐(1→6)‐β‐d ‐glucopyranose) of specific flavonoid glycosides such as rutin (quercetin 3‐O‐rutinoside). Microbial rutinosidases are part of the rutin catabolic pathway, enabling the microorganism to utilize rutin and related plant phenolic glycosides. Here, we report the first three‐dimensional structure of a rutinosidase determined at 1.27‐Å resolution. The rutinosidase from Aspergillus niger K2 (An Rut), a member of glycoside hydrolase family GH‐5, subfamily 23, was heterologously produced in Pichia pastoris . The X‐ray structure of An Rut is represented by a distorted (β/α)8 barrel fold with its closest structural homologue being an exo‐β‐(1,3)‐glucanase from Candida albicans (Ca Exg). The catalytic site is located in a deep pocket with a striking structural similarity to Ca Exg. However, the entrance to the active site of An Rut has been found to be different from that of Ca Exg – a mostly unstructured section of ~ 40 residues present in Ca Exg is missing in An Rut, whereas an additional loop of 13 amino acids partially covers the active site of An Rut. NMR analysis of reaction products provided clear evidence for a retaining reaction mechanism of An Rut. Unexpectedly, quercetin 3‐O‐glucoside was found to be a better substrate than rutin, and thus, An Rut cannot be considered a typical diglycosidase. Mutational analysis of conserved active site residues in combination with in silico modeling allowed identification of essential interactions for enzyme activity and helped to reveal further details of substrate binding. The protein sequence of An Rut has been revised.

中文翻译:

黑曲霉的芸香糖苷酶:晶体结构和对酶活性的了解。

Rutinosidases(α--rhamnosyl-β- d葡糖苷酶)催化糖苷配基之间的糖苷键的裂解的二糖芸香糖(α--rhamnopyranosyl-(1→6)-β- d -glucopyranose)特定的类黄酮的糖苷,如芦丁(槲皮素3- O-芦丁苷)。微生物芦丁糖苷酶是芦丁分解代谢途径的一部分,使微生物能够利用芦丁和相关的植物酚类糖苷。在这里,我们报告了以1.27-Å分辨率测定的芸香糖苷酶的第一个三维结构。从rutinosidase黑曲霉K2(一种车辙),糖苷水解酶家族GH-5,亚家族23的构件,在被异源生产巴斯德毕赤酵母An Rut的X射线结构以扭曲的(β/α)8桶折叠表示,其最接近的结构同源物是来自白色念珠菌Ca Exg)的exo- β-(1,3)-葡聚糖酶。催化部位位于与Ca Exg惊人的结构相似性的深袋中。但是,已发现An Rut活性位点的入口与Ca Exg的入口不同– An Rut中缺少Ca Exg中存在的〜40个残基的大部分非结构化片段,而另外13个氨基酸部分环涵盖了车辙。反应产物的NMR分析为An Rut的保留反应机理提供了清晰的证据。出乎意料的是,槲皮素-3- ö葡萄糖苷被发现是比芦丁更好的底物,因此,一个车辙不能被认为是一个典型的diglycosidase。保守的活性位点残基的突变分析与计算机模拟相结合,可以鉴定酶活性的基本相互作用,并有助于揭示底物结合的更多细节。An Rut的蛋白质序列已被修改。
更新日期:2020-01-13
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