Graduate School of Science and Engineering, Faculty of Engineering, Yamagata University, Yonezawa, Japan.
Department of Food, Life and Environmental Sciences, Faculty of Agriculture, Yamagata University, Tsuruoka, Japan.
Proteins. 2024 Feb;92(2):236-245. doi: 10.1002/prot.26608. Epub 2023 Oct 11.
The subsequent biochemical and structural investigations of the purified recombinant α-l-rhamnosidase from Aspergillus oryzae expressed in Pichia pastoris, designated as rAoRhaA, were performed. The specific activity of the rAoRhaA wild-type was higher toward hesperidin and narirutin, where the l-rhamnose residue was α-1,6-linked to β-d-glucoside, than toward neohesperidin and naringin with an α-1,2-linkage to β-d-glucoside. However, no activity was detected toward quercitrin, myricitrin, and epimedin C. rAoRhaA kinetic analysis indicated that K values for neohesperidin, naringin, and rutin were lower compared to those for hesperidin and narirutin. k values for hesperidin and narirutin were higher than those for neohesperidin, naringin, and rutin. High catalytic efficiency (k /K ) toward hesperidin and narirutin was a result of a considerably high k value, while K values for hesperidin and narirutin were higher than those for naringin, neohesperidin, and rutin. The crystal structure of rAoRhaA revealed that the catalytic domain was represented by an (α/α) -barrel with the active site located in a deep cleft and two β-sheet domains were also present in the N- and C-terminal sites of the catalytic domain. Additionally, five asparagine-attached N-acetylglucosamine molecules were observed. The catalytic residues of AoRhaA were suggested to be Asp254 and Glu524, and their catalytic roles were confirmed by mutational studies of D254N and E524Q variants, which lost their activity completely. Notably, three aspartic acids (Asp117, Asp249, and Asp261) located at the catalytic pocket were replaced with asparagine. D117N variant showed reduced activity. D249N and D261N variants activities drastically decreased.
随后对在巴斯德毕赤酵母中表达的重组α-l-鼠李糖苷酶（命名为 rAoRhaA）进行了生化和结构研究。rAoRhaA 野生型对橙皮苷和柚皮苷的比活性更高，其中 l-鼠李糖残基与β-d-葡萄糖苷以α-1,6-键连接，而对新橙皮苷和柚皮苷以α-1,2-键连接与β-d-葡萄糖苷。然而，对槲皮苷、杨梅素和淫羊藿苷 C 没有检测到活性。rAoRhaA 的动力学分析表明，与橙皮苷和柚皮苷相比，新橙皮苷、柚皮苷和芦丁的 K 值较低。新橙皮苷、柚皮苷和芦丁的 k 值高于橙皮苷和柚皮苷。对橙皮苷和柚皮苷具有较高的催化效率（k /K ）是由于 k 值相当高，而橙皮苷和柚皮苷的 K 值高于柚皮苷、新橙皮苷和芦丁。rAoRhaA 的晶体结构表明，催化结构域由（α/α）-桶代表，活性位点位于深裂缝中，两个β-折叠结构域也存在于催化结构域的 N-和 C-末端。此外，还观察到五个天冬酰胺连接的 N-乙酰葡萄糖胺分子。AoRhaA 的催化残基被推测为 Asp254 和 Glu524，通过 D254N 和 E524Q 变体的突变研究证实了它们的催化作用，这些变体完全失去了活性。值得注意的是，位于催化口袋的三个天冬氨酸（Asp117、Asp249 和 Asp261）被天冬酰胺取代。D117N 变体的活性降低。D249N 和 D261N 变体的活性急剧下降。
