Molecular Analyses of Starch-debranching Enzymes involved in Amylopectin Biosynthesis

淀粉脱支酶水解支链淀粉、糖原、支链淀粉和其他含有分支点的葡聚糖的α-1，6-糖苷键。这些酶通常分为两类，异淀粉酶和支链淀粉酶。在植物中，异淀粉酶由糖基因编码，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。本研究对水稻和马铃薯两种植物普鲁兰酶进行了纯化和性质研究，并解析了克雷伯氏菌普鲁兰酶的晶体结构。1.水稻胚乳普鲁兰酶：从水稻（Oryza sativa L.cv.Fujihikari）胚乳中分离纯化了淀粉脱支酶（R-酶或普鲁兰酶）。该酶的最适pH为5.5 ~ 7.5，最适温度为50 ℃左右。普鲁兰多糖的动力学参数，如Km和kcat，与克雷伯氏菌普鲁兰酶的动力学参数非常接近，但使用不同麦芽寡糖的抑制实验表明，普鲁兰多糖的动力学参数，如Km和kcat，与克雷伯氏菌普鲁兰酶的动力学参数非常接近，但使用不同麦芽寡糖的抑制实验表明，不同麦芽寡糖的动力学参数，如Km和kcat，与克雷伯氏菌普鲁兰酶的动力学参数非常接近，但使用不同麦芽寡糖的抑制实验表明，不同麦芽寡糖的动力学参数，如Km和kcat，与克雷伯氏菌普鲁兰酶的动力学参数非常接近，但不同麦芽寡糖的抑制实验表明，不同麦芽寡糖的动力学参数，如Km和kcat，与克雷伯氏菌普鲁兰酶的动力学参数非常接近。 ...更多信息 两种酶的活性位点2.马铃薯块茎普鲁兰酶：使用各种层析柱从马铃薯块茎中纯化该酶。该酶经SDS-PAGE测得分子量为100 kDa，凝胶过滤测得分子量为320 kDa，表明该酶具有三聚体或四聚体结构。该酶的最适pH为5.5 ~ 6.5，最适温度为40 ℃左右。该酶的米氏常数与水稻普鲁兰酶的米氏常数基本相同，而kcat不到水稻普鲁兰酶的十分之一。京都大学，我们已经通过使用SPring 8的同步辐射源以约1.7-1.9 A的分辨率改进了来自肺炎克雷伯氏菌的细菌普鲁兰酶及其与各种麦芽寡糖的复合物的晶体结构。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，而N3、A和C结构域与假单胞菌异淀粉酶的结构域仅具有弱相似性。N1结构域是一种新型的碳水化合物结合结构域，在CAZy数据库中被归类为CBM 41。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