Debranching enzymes of potato tubers (Solanum tuberosum L.). II. Purification of a pullulanase (R-enzyme) from potato tubers and comparison of its properties with those of the potato isoamylase.

Ishizaki, Yukuo; Taniguchi, Hajime; Maruyama, Yoshiharu; Nakamura, Mlchinori

doi:10.5458/jag1972.30.19

Cited by 13 publications

(7 citation statements)

References 2 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Mutation of ISA2 eliminates the same enzyme activity as mutation of ISA1, and the mutant phenotypes are indistinguishable from each other (Delatte et al, 2005;Wattebled et al, 2005). This is consistent with previous observations that isoamylases exist as multimeric enzymes (Ishizaki et al, 1983;Fujita et al, 1999;Dauvillé e et al, 2001), which in potato and rice have been shown to contain both ISA1 and ISA2 (Bustos et al, 2004;Utsumi and Nakamura, 2006). The loss of ISA3 or LDA alone does not lead to phytoglycogen production (Wattebled et al, 2005;Delatte et al, 2006).…”

Section: Introductionsupporting

confidence: 79%

Starch Granule Biosynthesis in Arabidopsis Is Abolished by Removal of All Debranching Enzymes but Restored by the Subsequent Removal of an Endoamylase

et al. 2008

View full text Add to dashboard Cite

Several studies have suggested that debranching enzymes (DBEs) are involved in the biosynthesis of amylopectin, the major constituent of starch granules. Our systematic analysis of all DBE mutants of Arabidopsis thaliana demonstrates that when any DBE activity remains, starch granules are still synthesized, albeit with altered amylopectin structure. Quadruple mutants lacking all four DBE proteins (Isoamylase1 [ISA1], ISA2, and ISA3, and Limit-Dextrinase) are devoid of starch granules and instead accumulate highly branched glucans, distinct from amylopectin and from previously described phytoglycogen. A fraction of these glucans are present as discrete, insoluble, nanometer-scale particles, but the structure and properties of this material are radically altered compared with wild-type amylopectin. Superficially, these data support the hypothesis that debranching is required for amylopectin synthesis. However, our analyses show that soluble glucans in the quadruple DBE mutant are degraded by a-and b-amylases during periods of net accumulation, giving rise to maltose and branched malto-oligosaccharides. The additional loss of the chloroplastic a-amylase AMY3 partially reverts the phenotype of the quadruple DBE mutant, restoring starch granule biosynthesis. We propose that DBEs function in normal amylopectin synthesis by promoting amylopectin crystallization but conclude that they are not mandatory for starch granule synthesis.

show abstract

Section: Introductionsupporting

confidence: 79%

Starch Granule Biosynthesis in Arabidopsis Is Abolished by Removal of All Debranching Enzymes but Restored by the Subsequent Removal of an Endoamylase

et al. 2008

View full text Add to dashboard Cite

show abstract

“…rMePUL exhibited the highest activity in acetate buffer pH at 6.0 (Figure like rMeISA3. The activity dropped by up to 50% at pH 5.0 and 7.0, with a sharp drop of activity below 5 or above 7, similar to the reported optimum pH of isoamylases from potato and maize (Ishizaki et al, 1983;Doehlert and Knutson, 1991;Rahman et al, 1998). Nevertheless, both optimal pH and temperature between MePUL and StPUL were similarly (Ishizaki et al, 1983).…”

Section: Discussionsupporting

confidence: 84%

“…Unlike other debranching enzymes from cassava, the MeISA1 and MeISA2 essentially exist as catalytically active hetermeric enzymes with various molar ratios (Panpetch et al, 2018b), while MeISA3 is a monomeric enzyme (Panpetch et al, 2018a). Additionally, SoPUL was reported as a monomeric protein (Renz et al, 1998) and partially purified StPUL from potato tuber present as a dimer analysed under nondenaturing condition however 3 protein bands were shown on SDS-PAGE gel (Ishizaki et al, 1983).…”

Section: Discussionmentioning

confidence: 98%

“…Here is less data for PUL, especially from dicotyledonous plants. Only partially purified PUL from potato (Solarium tuberosum L.) (StPUL) (Ishizaki et al, 1983) and purified PUL from spinach (Spinacia oleracea L.) (SoPUL) (Renz et al, 1998) were biochemically characterised to date. Moreover, the PUL from cassava, a tropical dicotyledonous plant, has not been studied before (Tappiban et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Cassava pullulanase and its synergistic debranching action with isoamylase 3 in starch catabolism

et al. 2023

View full text Add to dashboard Cite

Pullulanase (EC 3.2.1.41, PUL), a debranching enzyme belonging to glycoside hydrolase family 13 subfamily 13, catalyses the cleavage of α-1,6 linkages of pullulan and β-limit dextrin. The present work studied PUL from cassava Manihot esculenta Crantz (MePUL) tubers, an important economic crop. The Mepul gene was successfully cloned and expressed in E. coli and rMePUL was biochemically characterised. MePUL was present as monomer and homodimer, as judged by apparent mass of ~ 84 - 197 kDa by gel permeation chromatography analysis. Optimal pH and temperature were at pH 6.0 and 50 °C, and enzyme activity was enhanced by the addition of Ca2+ ions. Pullulan is the most favourable substrate for rMePUL, followed by β-limit dextrin. Additionally, maltooligosaccharides were potential allosteric modulators of rMePUL. Interestingly, short-chain maltooligosaccharides (DP 2 - 4) were significantly revealed at a higher level when rMePUL was mixed with cassava isoamylase 3 (rMeISA3), compared to that of each single enzyme reaction. This suggests that MePUL and MeISA3 debranch β-limit dextrin in a synergistic manner, which represents a major starch catabolising process in dicots. Additionally, subcellular localisation suggested the involvement of MePUL in starch catabolism, which normally takes place in plastids.

show abstract

“…The involvement of a debranching enzyme is therefore essential for its complete breakdown. Two classes of debranching enzyme, isoamylase (EC 3.2.1.68) and limit dextrinase (pullulanase‐type or R‐enzyme; EC 3.2.1.142), have been identified in plants (Ishizaki et al ., 1983; Doehlert & Knutson, 1991; Zhu et al ., 1998) and localized in chloroplasts (Okita et al ., 1979; Ludwig et al ., 1984; Kakefuda et al ., 1986; Li et al ., 1992a; Zeeman et al ., 1998a). Both are hydrolases and, while similar in amino acid sequence and predicted structure, differ in their substrate preference.…”

Section: Debranching Of Branched Glucansmentioning

confidence: 99%

The breakdown of starch in leaves

2004

View full text Add to dashboard Cite

Contents Summary Introduction Structure of the starch granule Initial attack on the granule and the role of glucan, water dikinase Debranching of branched glucans The metabolism of linear glucans Export of starch catabolites Metabolism of glucose and maltose The emerging pathway of starch breakdown and its regulation Acknowledgements References Summary This review describes recent progress in discovering the pathway of starch breakdown in leaves. The synthesis of starch from photo‐assimilated carbon is one of the major biochemical fluxes in plants. Despite this, the pathway through which this starch is remobilized has not been defined. Numerous enzymes that could participate in starch breakdown are present in leaves, but until recently, the relative importance of each had not been determined. Through studies using model species such as Arabidopsis and potato, significant progress has now been made in determining the roles of known enzymes, and in the discovery of novel proteins necessary for breakdown. These data allow a tentative pathway for starch breakdown to be mapped out, involving hydrolysis primarily to maltose and subsequent maltose export to the cytosol. This provides a framework for complete discovery of the pathway and for the analysis of its regulation.

show abstract

Debranching enzymes of potato tubers (Solanum tuberosum L.). II. Purification of a pullulanase (R-enzyme) from potato tubers and comparison of its properties with those of the potato isoamylase.

Cited by 13 publications

References 2 publications

Starch Granule Biosynthesis in Arabidopsis Is Abolished by Removal of All Debranching Enzymes but Restored by the Subsequent Removal of an Endoamylase

Starch Granule Biosynthesis in Arabidopsis Is Abolished by Removal of All Debranching Enzymes but Restored by the Subsequent Removal of an Endoamylase

Cassava pullulanase and its synergistic debranching action with isoamylase 3 in starch catabolism

The breakdown of starch in leaves

Contact Info

Product

Resources

About