Molecular Analyses of Starch-debranching Enzymes involved in Amylopectin Biosynthesis

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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