Molecular Analyses of Starch-debranching Enzymes involved in Amylopectin Biosynthesis

淀粉去分支酶水解支链淀粉、糖原、普鲁兰等含有分支点的葡聚糖的α-1,6-糖苷链。这些酶通常分为两类，异淀粉酶和普鲁兰酶。在植物中，异淀粉酶编码于糖基因中，被认为参与支链淀粉的生物合成，而普鲁兰酶的作用尚不清楚。在本研究中，我们对水稻和马铃薯两种植物普鲁兰酶进行了纯化和表征，并解决了克雷伯菌普鲁兰酶的晶体结构。水稻胚乳普鲁兰酶：从水稻（Oryza sativa L.cv.Fujihikari）发育中的胚乳中纯化出淀粉脱分枝酶（r -酶或普鲁兰酶）。酶的最适pH为5.5 ~ 7.5，最适温度为50℃左右。普鲁兰的动力学参数Km和kcat与克雷伯氏菌普鲁兰酶的动力学参数非常接近，但不同麦芽糖的抑制实验表明，两种酶的活性位点存在一定差异。马铃薯块茎普鲁兰酶：采用多种色谱柱从马铃薯块茎中纯化得到该酶。SDS-PAGE测定该酶分子量为100 kDa，凝胶过滤测定分子量为320 kDa，表明该酶具有三聚体或四聚体结构。酶的最适pH为5.5 ~ 6.5，最适温度为40℃左右。该酶的米切里斯常数与水稻普鲁兰酶基本相同，而kcat不到水稻酶的十分之一。普氏克雷伯菌普鲁兰酶：我们与京都大学的Mikami等人合作，利用SPring8的同步辐射源，以1.7-1.9 A的分辨率精细了来自肺炎克雷伯菌及其与各种低聚麦芽糖复合物的细菌普鲁兰酶的晶体结构。该酶由5个结构域（N1,N2,N3，A和C）组成。N1和N2结构域是克雷伯氏菌普鲁兰酶的特征结构域，而N3、A和C结构域与假单胞菌异淀粉酶的结构域只有微弱的相似性。在CAZy数据库中发现N1结构域是一种新的碳水化合物结合结构域，属于CBM41。少

Starch debranching enzymes hydrolyze α-1,6-glucosidic linkage of amylopectin, glycogen, pullulan and other glucan containing branching points. These enzymes are usually classified to two categories, isoamylase and pullulanase. In plants, isoamylase is coded in sugary gene and considered to be involved in amylopectin biosynthesis, whereas the role of pullulanase is not known. In this study, we purified and characterized two plant pullulanases, rice and potato, and have solved the crystal structures of Klebsiella pullulanase.1.Rice endosperm pullulanase : Starch debranching enzyme (R-enzyme or pullulanase) was purified to homogeneity from developing endosperm of rice (Oryza sativa L.cv.Fujihikari). The optimum pH of the enzyme was 5.5 to 7.5 and the optimum temperature was around 50C. Kinetic parameters, such as Km and kcat, for pullulan were very close to those of Klebsiella pullulanase, but the inhibition experiments using various maltooligosaccharides indicates some differences in the … More active sites of two enzymes.2.Potato tuber pullulanase : The enzyme was purified from potato tuber using various chromatography columns. Molecular weight of this enzyme was 100 kDa by SDS-PAGE and 320 kDa by gel filtration, which indicates that potato pullulanase has trimeric or tetrameric structure. The optimum pH of the enzyme was 5.5 to 6.5 and the optimum temperature was around 40C. Michaelis constant of this enzyme was almost the same as rice pullulanase, while the kcat was less than one tenth of rice enzyme.3.Klebsiella pullulanase : In collaboration with Mikami et al., Kyoto University, we have refined the crystal structures of bacterial pullulanase derive from Klebsiella pneulnoniae and its complexes with various maltooligosaccharides at around 1.7-1.9 A resolution by using a synchrotron radiation source at SPring8. This enzyme is composed of 5 domains (N1,N2,N3,A, and C). The N1 and N2 domains are characteristic of Klebsiella pullulanase, while N3,A, and C domains have only weak similarities with those of Pseudomonas isoamylase. The N1 domain was found to be a new type of carbohydrate-binding domain classified to CBM41 in CAZy database. Less