Plant Starch Metabolism Research Articles

Raw starch degrading α-amylases: an unsolved riddle

AbstractStarch is an important food ingredient and a substrate for the production of many industrial products. Biological and industrial processes involve hydrolysis of raw starch, such as digestion by humans and animals, starch metabolism in plants, and industrial starch conversion for obtaining glucose, fructose and maltose syrup or bioethanol. Raw starch degrading α-amylases (RSDA) can directly degrade raw starch below the gelatinization temperature of starch. Knowledge of the structures and properties of starch and RSDA has increased significantly in recent years. Understanding the relationships between structural peculiarities and properties of RSDA is a prerequisite for efficient application in different aspects of human benefit from health to the industry. This review summarizes recent advances on RSDA research with emphasizes on representatives of glycoside hydrolase family GH13. Definite understanding of raw starch digesting ability is yet to come with accumulating structural and functional studies of RSDA.

Assessing the Biological Activity of the Glucan Phosphatase Laforin.

Glucan phosphatases are a recently discovered family of enzymes that dephosphorylate either starch or glycogen and are essential for proper starch metabolism in plants and glycogen metabolism in humans. Mutations in the gene encoding the only human glucan phosphatase, laforin, result in the fatal, neurodegenerative, epilepsy known as Lafora disease. Here, we describe phosphatase assays to assess both generic laforin phosphatase activity and laforin's unique glycogen phosphatase activity.

Expression and characterization of 4-α-glucanotransferase genes from Manihot esculenta Crantz and Arabidopsis thaliana and their use for the production of cycloamyloses

4-α-Glucanotransferase or disproportionating enzyme (D-enzyme, DPE) catalyzes the α-1.4 glycosyl transfer between oligosaccharides. Type I D-enzyme (DPE1) can transfer maltosyl unit from one 1.4-α-d-glucan to an acceptor mono- or oligo-saccharide, which reflects the physiological role of DPE1 in plant starch metabolism. In this study, the genes encoding DPE1 from Arabidopsis thaliana (AtDPE1) and Manihot esculenta Crantz cultivar KU50 (MeDPE1) were cloned and expressed in Escherichia coli and purified to homogeneity. MeDPE1 encoded 585 amino acid residues, including a 56 residue signal peptide, while AtDPE1 encoded 576 amino acid residues with a 45 residue signal peptide. The molecular mass of both mature enzymes, estimated from deduced amino acid sequence, were the same at 59.4kDa, with a pI of 5.13. The predicted structures of both enzymes showed the conserved 250's loop and three catalytic amino acid residues, characteristics of disproportionating enzymes in the GH77 glycoside hydrolase family. Biochemical characterization showed that both purified recombinant enzymes were homodimers in solution, with similar optimum pH and temperature for disproportionating activity at pH 6–8 and 37°C. Using potato amylose as a substrate, AtDPE1 can produce cycloamyloses in the range 16–50 glucose residues, while products from the action of MeDPE1 on the same substrate were in a wider range of 16 to DP>60. These recombinant enzymes are useful tools for elucidation of their functional roles in starch metabolism and for applications in the starch industry.

Hyperphosphorylation of Glucosyl C6 Carbons and Altered Structure of Glycogen in the Neurodegenerative Epilepsy Lafora Disease

Laforin or malin deficiency causes Lafora disease, characterized by altered glycogen metabolism and teenage-onset neurodegeneration with intractable and invariably fatal epilepsy. Plant starches possess small amounts of metabolically essential monophosphate esters. Glycogen contains similar phosphate amounts, which are thought to originate from a glycogen synthase error side reaction and therefore lack any specific function. Glycogen is also believed to lack monophosphates at glucosyl carbon C6, an essential phosphorylation site in plant starch metabolism. We now show that glycogen phosphorylation is not due to a glycogen synthase side reaction, that C6 is a major glycogen phosphorylation site, and that C6 monophosphates predominate near centers of glycogen molecules and positively correlate with glycogen chain lengths. Laforin or malin deficiency causes C6 hyperphosphorylation, which results in malformed long-chained glycogen that accumulates in many tissues, causing neurodegeneration in brain. Our work advances the understanding of Lafora disease pathogenesis and suggests that glycogen phosphorylation has important metabolic function.

Lafora disease: Convergence of neurodegeneration with plant starch metabolism

Reversible phosphorylation modulates many proteins involved in glycogenesis and glycogenolysis. Recent results from mammalian cells, mouse models, eukaryotic algae, and plants have identified a new phosphorylation target in glycogen metabolism, the glucan itself.Mutations in EPM2A lead to Lafora disease, a fatal neurodegenerative disease. EPM2A encodes laforin, a bimodular protein with an amino‐terminal carbohydrate binding module and a carboxy‐terminal dual specificity phosphatase domain. We recently demonstrated that laforin removes phosphate from phosphoglucans.Starch Excess 4 (SEX4) encodes a similar phosphatase in plants, and we demonstrated that laforin and SEX4 are the founding members of the glucan phosphatases. Multiple groups have now shown that these phosphatases dephosphorylate phosphoglucans, but the physical basis for the function of laforin and SEX4 is unclear.We determined the crystal structure of SEX4, and found that it contains a phosphatase domain (DSP), carbohydrate binding module (CBM), and a previously unknown carboxy‐terminal domain. Hydrogen‐deuterium exchange mass spectrometry (DXMS) identified regions of the DSP domain that interact with glucans. Cumulatively, these results reveal the unique tertiary architecture of SEX4 that provides the physical basis for its function as a glucan phosphatase and present a model for the function of laforin.

The Role of D-enzyme in Starch Metabolism in Plants

D-enzyme (EC 2.4.1.25) is believed to be involved in starch metabolism, but the function in vivo is not known. In order to investigate the role of D-enzyme, several approaches have been undertaken. A biochemical analysis of the purified potato D-enzyme suggested that high molecular weight starch (amylose and amylopectin) can serve as donor and acceptor, and very long α-1, 4-glucans or even highly branched glucans can be transferred by the enzyme. Transgenic potato plants with dramatically reduced D-enzyme activity were obtained by introducing sense and antisense D-enzyme cDNA sequences with the appropriate promoter sequence. The tubers from these plants sprouted later and the growth of sprouts was slower than the wild type. However, no significant difference was found in starch produced in tubers, either in its quantity or quality. From these results, the possible role of Denzyme in starch metabolism is discussed.

Starch-binding domains in the post-genome era.

Starch belongs to the most abundant biopolymers on Earth. As a source of energy, starch is degraded by a large number of various amylolytic enzymes. However, only about 10% of them are capable of binding and degrading raw starch. These enzymes usually possess a distinct sequence-structural module, the so-called starchbinding domain (SBD). In general, all carbohydrate-binding modules (CBMs) have been classified into the CBM families. In this sequence-based classification the individual types of SBDs have been placed into seven CBM families: CBM20, CBM21, CBM25, CBM26, CBM34, CBM41 and CBM45. The family CBM20, known also as a classical C-terminal SBD of microbial amylases, is the most thoroughly studied. The three-dimensional structures have already been determined by X-ray crystallography or nuclear magnetic resonance for SBDs from five CBM families (20, 25, 26, 34 and 41), and the structure of the CBM21 has been modelled. Despite differences among the amino acid sequences, the fold of a distorted beta-barrel seems to be conserved together with a similar way of substrate binding (mainly stacking interactions between aromatic residues and glucose rings). SBDs have recently been discovered in many non-amylolytic proteins. These may, for example, have regulatory functions in starch metabolism in plants or glycogen metabolism in mammals. SBDs have also found practical uses.

Phosphatase as Starch Control

Plant metabolism shifts dramatically with the daily light cycle, from a situation in which carbon is fixed into sugars and stored as starch during photosynthesis to one in which reserves of starch are used for energy during the night. Precisely how this switch from biosynthesis to consumption is regulated has not been clear. But now Sokolov et al. have characterized an enzyme that may couple metabolism of starch to daily cycles. Taking a cue from the knowledge that in animals, accumulation of glycogen is regulated by a dual-specificity phosphatase (that is, one that modifies both phosphotyrosine and phosphoserine or phosphothreonine residues), the authors characterized a related phosphatase that they call DSP4. Mutant Arabidopsis plants in which the DSP4 gene was altered showed increased accumulation of starch in the leaves. DSP4 also interacted directly with starch granules in in vitro binding assays and, in plants expressing DSP4 tagged with green fluorescent protein, DSP4 was associated with starch granules during the day but not at night. The pH of the chloroplast stroma increases from about pH7 at night to about pH8 in the daytime. The redox environment also changes, with various enzymes being reduced during the day. The authors propose that these signals may couple to control of starch metabolism through DSP4 because DSP4 activity in in vitro assays was lost as pH increased from pH7 to pH8. Addition of oxidants decreased binding of DSP4 to starch granules and decreased the activity of the phosphatase. Thus, plants may have a phosphorylation-mediated mechanism controlling storage of energy in starch that resembles that of animals for control of glycogen metabolism. A related paper recently appeared in the Journal of Biological Chemistry . L. N. Sokolov, J. R. Dominguez-Solis, A.-L. Allary, B. B. Buchanan, S. Luan, A redox-regulated chloroplast protein phosphatase binds to starch diurnally and functions in its accumulation. Proc. Natl. Acad. Sci. U.S.A. 103 , 9732-9737 (2006). [Abstract] [Full Text] T. Niittylä, S. Comparot-Moss, W.-L. Lue, G. Messerli, M. Trevisan, M. D. J. Seymour, J. A. Gatehouse, D. Villadsen, S. M. Smith, J. Chen, S. C. Zeeman, A. M. Smith, Similar protein phosphatases control starch metabolism in plants and glycogen metabolism in mammals. J. Biol. Chem. 281 , 11815-11818 (2006). [Abstract] [Full Text]

Similar Protein Phosphatases Control Starch Metabolism in Plants and Glycogen Metabolism in Mammals

We report that protein phosphorylation is involved in the control of starch metabolism in Arabidopsis leaves at night. sex4 (starch excess 4) mutants, which have strongly reduced rates of starch metabolism, lack a protein predicted to be a dual specificity protein phosphatase. We have shown that this protein is chloroplastic and can bind to glucans and have presented evidence that it acts to regulate the initial steps of starch degradation at the granule surface. Remarkably, the most closely related protein to SEX4 outside the plant kingdom is laforin, a glucan-binding protein phosphatase required for the metabolism of the mammalian storage carbohydrate glycogen and implicated in a severe form of epilepsy (Lafora disease) in humans.

A Novel Thermoreversible Gelling Product Made by Enzymatic Modification of Starch

Amylomaltases or D-enzyme (4-α-glucanotransferases; E.C. 2.4.1.25) are carbohydrate-active enzymes that catalyze the transfer of glucan units from one α-glucan to another in a disproportionation reaction. These enzymes are involved in starch metabolism in plants or maltose/glycogen metabolism in many microorganisms. The amylomaltase of the hyperthermophilic bacterium Thermus thermophilus HB8 was overproduced in Escherichia coli, partially purified and used to modify potato starch. The action of amylomaltase caused the disappearance of amylose and the broadening of the side-chain length distribution in amylopectin, which resulted in a product with both shorter and longer side chains than in the parent starch. Amylomaltase-treated potato starch showed thermoreversible gelation at concentrations of 3% (w/v) or more, thus making it comparable to gelatin. Because of its animal origin, gelatin is not accepted by several consumer groups. Therefore, the amylomaltase-treated potato starch might be a good plant-derived substitute for gelatin.

From bacterial glycogen to starch: understanding the biogenesis of the plant starch granule.

Plants, green algae, and cyanobacteria synthesize storage polysaccharides by a similar ADPglucose-based pathway. Plant starch metabolism can be distinguished from that of bacterial glycogen by the presence of multiple forms of enzyme activities for each step of the pathway. This multiplicity does not coincide with any functional redundancy, as each form has seemingly acquired a distinctive and conserved role in starch metabolism. Comparisons of phenotypes generated by debranching enzyme-defective mutants in Escherichia coli and plants suggest that enzymes previously thought to be involved in polysaccharide degradation have been recruited during evolution to serve a particular purpose in starch biosynthesis. Speculations have been made that link this recruitment to the appearance of semicrystalline starch in photosynthetic eukaryotes. Besides the common core pathway, other enzymes of malto-oligosaccharide metabolism are required for normal starch metabolism. However, according to the genetic and physiological system under study, these enzymes may have acquired distinctive roles.

Defining the Functions of Maltodextrin Active Enzymes in Starch Metabolism in the Unicellular Alga Chlamydomonas reinhardtii

Bacterial glycogen and plant starch metabolism both require the presence of malto-oligosaccharide assimilation enzymes. In Escherichia coil maltotetraose is generated through debranching of the glycogen limit dextrin produced by glycogen phosphorylase. This maltotetraose if further metabolised through the combined action of amylomaltase (an α-1, 4 glucanotransferase) and maltodextrin phosphorylase. In the starch accumulating alga Chlamydomonas reinhardtii we show that a deficiency in D-enzyme (the plant α-1, 4 glucanotransferase) leads to a severe decrease in starch content and a modification in amylopectin structure as well as a modification in amylose content. We further show that there are 2 distinct plastidial phosphorylases in Chlamydomonas. Kinetic and genetic studies suggest these forms may be related to the maltodextrin and glycogen-type of phosphorylases from bacteria.

Impact of water stress on yield and quality of cassava starch

Cassava ( Manihot esculenta Crantz) is an important source of industrial raw materials. Products obtained from cassava include chip/pellets for animal feed and starch. Important for major industrial uses are the amount and quality of starch obtained from this crop. Production efficiency, including yield and quality of starch, from cassava is markedly influenced by environmental conditions, especially water stress during early plant development and immediately before root harvest. In early plant development plants deprived of water for the first 6 months were characterized by a lower yield of starch compared to plants without water stress (starch yields of six varieties including Rayong 1, Rayong 5, Rayong 60, Rayong 90, Kasetsart 50 and CMR 33-57-81 were 0.1–0.2 and 5.0–8.7 t/ha for water-stressed and without water-stressed plants). Furthermore, starch from plants deprived of water for the first 6 months of growth, was functionally different to that laid-down under optimum growing conditions. Plants responded to subsequent rainfall and after 2 months contained significant amounts of starch, though this amount was less than was expected. Despite the fact that water-stressed plants responded to the availability of water by producing starch, most functional properties remained different. The portfolio of changes was sufficient as regards the starch of lower quality. Most effected were the hydration properties. Starch granules despite being smaller (mean size and distribution) than expected were morphologically normal. A second drought period further influenced some of the starch properties, but the sustained influence of the early drought seemed to dominate the response of the plant's starch metabolism. All varieties were similarly affected.

Cyclic Glucans Produced by the Intramolecular Transglycosylation Activity of Potato D-Enzyme on Amylopectin

Potato D-enzyme catalyses an intramolecular transglycosylation reaction on amylose to produce cycloamylose, a novel cyclic α-1,4 glucan. To determine if a similar activity could be observed with a high molecular weight branched substrate, recombinant potato D-enzyme was incubated with amylopectin. The substrate was converted into two fractions of lower molecular mass. Fraction I comprised 15% cyclic molecules of which the majority contained both α-1,4 and α-1,6 links. These were shown to be branched molecules with branches shorter than those in amylopectin. Fraction II comprised 80% cyclic molecules of which the majority contained only α-1,4 links (cycloamylose). Since fraction II appeared before fraction I, we propose that D-enzyme first catalysed the cyclisation of the outer side chains of amylopectin and then the cyclisation of inner chains to produce branched clusters. These results demonstrate that D-enzyme can catalyse the transfer of branched glucans, and suggest novel ways in which it may participate in starch metabolism in plants.