Mechanistic Insights into Glucan Phosphatase Activity against Polyglucan Substrates

Glucan phosphatases are central to the regulation of starch and glycogen metabolism. Plants contain two known glucan phosphatases, Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), which dephosphorylate starch. Starch is water-insoluble and reversible phosphorylation solubilizes its outer surface allowing processive degradation. Vertebrates contain a single known glucan phosphatase, laforin, that dephosphorylates glycogen. In the absence of laforin, water-soluble glycogen becomes insoluble, leading to the neurodegenerative disorder Lafora Disease. Because of their essential role in starch and glycogen metabolism glucan phosphatases are of significant interest, yet a comparative analysis of their activities against diverse glucan substrates has not been established. We identify active site residues required for specific glucan dephosphorylation, defining a glucan phosphatase signature motif (CζAGΨGR) in the active site loop. We further explore the basis for phosphate position-specific activity of these enzymes and determine that their diverse phosphate position-specific activity is governed by the phosphatase domain. In addition, we find key differences in glucan phosphatase activity toward soluble and insoluble polyglucan substrates, resulting from the participation of ancillary glucan-binding domains. Together, these data provide fundamental insights into the specific activity of glucan phosphatases against diverse polyglucan substrates.     

Structure of the Arabidopsis Glucan Phosphatase LIKE SEX FOUR2 Reveals a Unique Mechanism for Starch Dephosphorylation

Starch is a water-insoluble, Glc-based biopolymer that is used for energy storage and is synthesized and degraded in a diurnal manner in plant leaves. Reversible phosphorylation is the only known natural starch modification and is required for starch degradation in planta. Critical to starch energy release is the activity of glucan phosphatases; however, the structural basis of dephosphorylation by glucan phosphatases is unknown. Here, we describe the structure of the Arabidopsis thaliana starch glucan phosphatase LIKE SEX FOUR2 (LSF2) both with and without phospho-glucan product bound at 2.3Å and 1.65Å, respectively. LSF2 binds maltohexaose-phosphate using an aromatic channel within an extended phosphatase active site and positions maltohexaose in a C3-specific orientation, which we show is critical for the specific glucan phosphatase activity of LSF2 toward native Arabidopsis starch. However, unlike other starch binding enzymes, LSF2 does not possess a carbohydrate binding module domain. Instead we identify two additional glucan binding sites located within the core LSF2 phosphatase domain. This structure is the first of a glucan-bound glucan phosphatase and provides new insights into the molecular basis of this agriculturally and industrially relevant enzyme family as well as the unique mechanism of LSF2 catalysis, substrate specificity, and interaction with starch granules.     

EARLY STARVATION1 specifically affects the phosphorylation action of starch‐related dikinases

Starch phosphorylation by starch‐related dikinases glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) is a key step in starch degradation. Little information is known about the precise structure of the glucan substrate utilized by the dikinases and about the mechanisms by which these structures may be influenced. A 50‐kDa starch‐binding protein named EARLY STARVATION1 (ESV1) was analyzed regarding its impact on starch phosphorylation. In various in vitro assays, the influences of the recombinant protein ESV1 on the actions of GWD and PWD on the surfaces of native starch granules were analyzed. In addition, we included starches from various sources as well as truncated forms of GWD. ESV1 preferentially binds to highly ordered, α‐glucans, such as starch and crystalline maltodextrins. Furthermore, ESV1 specifically influences the action of GWD and PWD at the starch granule surface. Starch phosphorylation by GWD is decreased in the presence of ESV1, whereas the action of PWD increases in the presence of ESV1. The unique alterations observed in starch phosphorylation by the two dikinases are discussed in regard to altered glucan structures at the starch granule surface.     

The phosphoglucan phosphatase like sex Four2 dephosphorylates starch at the C3-position in Arabidopsis

Starch contains phosphate covalently bound to the C6-position (70 to 80% of total bound phosphate) and the C3-position (20 to 30%) of the glucosyl residues of the amylopectin fraction. In plants, the transient phosphorylation of starch renders the granule surface more accessible to glucan hydrolyzing enzymes and is required for proper starch degradation. Phosphate also confers desired properties to starch-derived pastes for industrial applications. In Arabidopsis thaliana, the removal of phosphate by the glucan phosphatase Starch Excess4 (SEX4) is essential for starch breakdown. We identified a homolog of SEX4, LSF2 (Like Sex Four2), as a novel enzyme involved in starch metabolism in Arabidopsis chloroplasts. Unlike SEX4, LSF2 does not have a carbohydrate binding module. Nevertheless, it binds to starch and specifically hydrolyzes phosphate from the C3-position. As a consequence, lsf2 mutant starch has elevated levels of C3-bound phosphate. SEX4 can release phosphate from both the C6- and the C3-positions, resulting in partial functional overlap with LSF2. However, compared with sex4 single mutants, the lsf2 sex4 double mutants have a more severe starch-excess phenotype, impaired growth, and a further change in the proportion of C3- and C6-bound phosphate. These findings significantly advance our understanding of the metabolism of phosphate in starch and provide innovative options for tailoring novel starches with improved functionality for industry.     

The Laforin-Like Dual-Specificity Phosphatase SEX4 from Arabidopsis Hydrolyzes Both C6- and C3-Phosphate Esters Introduced by Starch-Related Dikinases and Thereby Affects Phase Transition of α-Glucans

The biochemical function of the Laforin-like dual-specific phosphatase AtSEX4 (EC 3.1.3.48) has been studied. Crystalline maltodextrins representing the A- or the B-type allomorph were prephosphorylated using recombinant glucan, water dikinase (StGWD) or the successive action of both plastidial dikinases (StGWD and AtPWD). AtSEX4 hydrolyzed carbon 6-phosphate esters from both the prephosphorylated A- and B-type allomorphs and the kinetic constants are similar. The phosphatase also acted on prelabeled carbon-3 esters from both crystalline maltodextrins. Similarly, native starch granules prelabeled in either the carbon-6 or carbon-3 position were also dephosphorylated by AtSEX4. The phosphatase did also hydrolyze phosphate esters of both prephosphorylated maltodextrins when the (phospho)glucans had been solubilized by heat treatment. Submillimolar concentrations of nonphosphorylated maltodextrins inhibited AtSEX4 provided they possessed a minimum of length and had been solubilized. As opposed to the soluble phosphomaltodextrins, the AtSEX4-mediated dephosphorylation of the insoluble substrates was incomplete and at least 50% of the phosphate esters were retained in the pelletable (phospho)glucans. The partial dephosphorylation of the insoluble glucans also strongly reduced the release of nonphosphorylated chains into solution. Presumably, this effect reflects fast structural changes that following dephosphorylation occur near the surface of the maltodextrin particles. A model is proposed defining distinct stages within the phosphorylation/dephosphorylation-dependent transition of α-glucans from the insoluble to the soluble state.The metabolism of starch, the most prominent storage carbohydrate in plants, is assumed to require approximately 30 to 40 distinct (iso)enzymes (Deschamps et al., 2008), but, presumably, the list of the starch-related proteins is not yet complete. Several novel proteins (and protein functions) essential for the normal starch metabolism have recently been identified among which are two α-glucan phosphorylating dikinases. One dikinase (glucan, water dikinase [GWD], EC 2.7.9.4) utilizes ATP as dual phosphate donor and esterifies the C6 position of amylopectin-related glucosyl residues, whereas the other dikinase (phosphoglucan, water dikinase [PWD], EC 2.7.9.5) selectively transfers the β-phosphate group from ATP to the C3 position of glucosyl residues (Ritte et al., 2006).Two other previously unknown starch-related enzymes were designated as SEX4 protein (EC 3.1.3.48; At3g52180; previous designations PTPKIS1 and DSP4) and as Like Sex Four1 (LSF1) protein (At3g01510; previously named PTPKIS2; Comparot-Moss et al., 2010). Both proteins are predicted to contain a noncatalytic carbohydrate-binding module (CBM; Boraston et al., 2004; Shoseyov et al., 2006) and a catalytic dual-specificity phosphatase (DSP) domain. The latter is shared by the large family of DSPs that dephosphorylate distinct target phosphoproteins both at phosphotyrosine and phosphoserine/phosphothreonine residues. Some DSPs also act on various nonproteinaceous substrates, such as phospholipids or phosphorylated polyglycans (Pulido and Hooft van Huijsduijnen, 2008).Arabidopsis (Arabidopsis thaliana) mutants lacking a functional SEX4 protein contain both elevated starch levels and significant amounts of soluble phosphooligoglucans that are below the limit of detection in wild-type plants and probably originate from starch. However, the precise biochemical function of SEX4 is far from being clear (Kötting et al., 2009). The phenotype of the SEX4-deficient mutant is complex: Transitory starch possesses an elevated amylose-to-amylopectin ratio but the phosphate content of amylopectin is not increased. It has been hypothesized that SEX4 and LSF1 selectively hydrolyze C6- and C3-phosphate esters, respectively, but experimental evidence is lacking (Kötting et al., 2009). Likewise, it is unknown whether SEX4 preferentially acts on particulate starch or on soluble phosphoglucans.Crystalline maltodextrins (MD_cryst) have recently been introduced as a model mimicking some structural features of the native starch granules (Hejazi et al., 2008). They can be crystallized as either the A- or the B-type allomorph (Gallant et al., 1997; Gérard et al., 2001). Recombinant StGWD phosphorylates both maltodextrin allomorphs with a far higher rate than native starch granules and thereby initiates solubilization of both phosphorylated and nonphosphorylated maltodextrins. In vitro both allomorphs act also as substrate for PWD provided a prephosphorylation by GWD (Hejazi et al., 2009).In this study, we used the prephosphorylated A- and B-type allomorphs of MD_cryst to study biochemical functions of AtSEX4. As highly ordered α-glucans are the preferred sites of the dikinase-mediated phosphorylation, we designed experiments to answer the following questions: Does SEX4 preferentially act on phosphorylated insoluble or soluble glucans? If insoluble α-glucans are the preferred substrate, does the phosphatase distinguish between the A- and the B-type allomorph? Does SEX4 preferentially or selectively hydrolyze C6-phosphate esters? Does SEX4 also interact with nonphosphorylated oligoglucans? Finally, assuming that SEX4 acts on insoluble phosphoglucans, does the removal of phosphate esters affect the phase transition and/or the physical order of the glucans?     

Conserved structure and varied expression reveal key roles of phosphoglucan phosphatase gene starch excess 4 in barley

As one of the phosphoglucan phosphatases, starch excess 4 (SEX4) encoded by SEX4 gene has recently been intensively studied because of its vital role in the degradation of leaf starch. In this study, we isolated and chromosomally mapped barley SEX4, characterized its gene and protein structure, predicted the cis-elements of its promoter, and analysed its expression based on real-time quantitative PCR and publically available microarray data. The full length of barely SEX4 (HvSEX4) was 4,598 bp and it was mapped on the long arm of chromosome 4H (4HL). This gene contained 14 exons and 13 introns in all but two of the species analysed, Arabidopsis (13 exons and 12 introns) and Oryza brachyantha (12 exons and 11 introns). An exon–intron junction composed of intron 4 to intron 7 and exon 5 to exon 8 was highly conserved among the analysed species. SEX4 is characterized with conserved functional domains (dual specificity phosphatase domain and carbohydrate-binding module 48) and varied chloroplast transit peptide and C-terminal. Expression analyses indicated that: (1) SEX4 was mainly expressed in anthers of barley, young leaf and anthers of rice, and leaf of Arabidopsis; (2) it exhibited a diurnal pattern in barley, rice and Arabidopsis; (3) significant difference in the expression of SEX4 was not detected for either barley or rice under any of the investigated stresses; and (4) it was significantly down-regulated at middle stage and up-regulated at late stage under cold treatment, down-regulated at early stage under heat treatment, and up-regulated at late stage under salt treatment in Arabidopsis. The strong relationships detected in the current study between SEX4 and glucan, water dikinases (GWD) or phosphoglucan, water dikinases (PWD) were discussed. Collectively, our results provide insights into genetic manipulation of SEX4, especially in monocotyledon and uncovering the possible roles of SEX4 in plant development.     

Degradation of Native Starch Granules by Barley alpha-Glucosidases

The initial hydrolysis of native (unboiled) starch granules in germinating cereal kernels is considered to be due to α-amylases. We report that barley (Hordeum vulgare L.) seed α-glucosidases (EC 3.2.1.20) can hydrolyze native starch granules isolated from barley kernels and can do so at rates comparable to those of the predominant α-amylase isozymes. Two α-glucosidase charge isoforms were used individually and in combination with purified barley α-amylases to study in vitro starch digestion. Dramatic synergism, as much as 10.7-fold, of native starch granule hydrolysis, as determined by reducing sugar production, occurred when high pl α-glucosidase was combined with either high or low pl α-amylase. Synergism was also found when low pl α-glucosidase was combined with α-amylases. Scanning electron micrographs revealed that starch granule degradation by α-amylases alone occurred specifically at the equatorial grooves of lenticular granules. Granules hydrolyzed by combinations of α-glucosidases and α-amylases exhibited larger and more numerous holes on granule surfaces than did those granules attacked by α-amylase alone. As the presence of α-glucosidases resulted in more areas being susceptible to hydrolysis, we propose that this synergism is due, in part, to the ability of the α-glucosidases to hydrolyze glucosidic bonds other than α-1,4- and α-1,6- that are present at the granule surface, thereby eliminating bonds which were barriers to hydrolysis by α-amylases. Since both α-glucosidase and α-amylase are synthesized in aleurone cells during germination and secreted to the endosperm, the synergism documented here may function in vivo as well as in vitro.     

Degradation of Glucan Primers in the Absence of Starch Synthase 4 Disrupts Starch Granule Initiation in Arabidopsis

Arabidopsis leaf chloroplasts typically contain five to seven semicrystalline starch granules. It is not understood how the synthesis of each granule is initiated or how starch granule number is determined within each chloroplast. An Arabidopsis mutant lacking the glucosyl-transferase, STARCH SYNTHASE 4 (SS4) is impaired in its ability to initiate starch granules; its chloroplasts rarely contain more than one large granule, and the plants have a pale appearance and reduced growth. Here we report that the chloroplastic α-amylase AMY3, a starch-degrading enzyme, interferes with granule initiation in the ss4 mutant background. The amy3 single mutant is similar in phenotype to the wild type under normal growth conditions, with comparable numbers of starch granules per chloroplast. Interestingly, the ss4 mutant displays a pleiotropic reduction in the activity of AMY3. Remarkably, complete abolition of AMY3 (in the amy3 ss4 double mutant) increases the number of starch granules produced in each chloroplast, suppresses the pale phenotype of ss4, and nearly restores normal growth. The amy3 mutation also restores starch synthesis in the ss3 ss4 double mutant, which lacks STARCH SYNTHASE 3 (SS3) in addition to SS4. The ss3 ss4 line is unable to initiate any starch granules and is thus starchless. We suggest that SS4 plays a key role in granule initiation, allowing it to proceed in a way that avoids premature degradation of primers by starch hydrolases, such as AMY3.     

Arabidopsis thaliana AMY3 Is a Unique Redox-regulated Chloroplastic α-Amylase

α-Amylases are glucan hydrolases that cleave α-1,4-glucosidic bonds in starch. In vascular plants, α-amylases can be classified into three subfamilies. Arabidopsis has one member of each subfamily. Among them, only AtAMY3 is localized in the chloroplast. We expressed and purified AtAMY3 from Escherichia coli and carried out a biochemical characterization of the protein to find factors that regulate its activity. Recombinant AtAMY3 was active toward both insoluble starch granules and soluble substrates, with a strong preference for β-limit dextrin over amylopectin. Activity was shown to be dependent on a conserved aspartic acid residue (Asp⁶⁶⁶), identified as the catalytic nucleophile in other plant α-amylases such as the barley AMY1. AtAMY3 released small linear and branched glucans from Arabidopsis starch granules, and the proportion of branched glucans increased after the predigestion of starch with a β-amylase. Optimal rates of starch digestion in vitro was achieved when both AtAMY3 and β-amylase activities were present, suggesting that the two enzymes work synergistically at the granule surface. We also found that AtAMY3 has unique properties among other characterized plant α-amylases, with a pH optimum of 7.5–8, appropriate for activity in the chloroplast stroma. AtAMY3 is also redox-regulated, and the inactive oxidized form of AtAMY3 could be reactivated by reduced thioredoxins. Site-directed mutagenesis combined with mass spectrometry analysis showed that a disulfide bridge between Cys⁴⁹⁹ and Cys⁵⁸⁷ is central to this regulation. This work provides new insights into how α-amylase activity may be regulated in the chloroplast.     

Dynamics of starch granule biogenesis – the role of redox-regulated enzymes and low-affinity carbohydrate-binding modules

Abstract

The deposition and degradation of starch in plants is subject to extensive post-translational regulation. To permit degradation of B-type crystallites present in tuberous and leaf starch these starch types are phosphorylated by glucan, water dikinase (GWD). At the level of post-translational redox regulation, ADPglucose pyrophosphorylase, β-amylase (BAM1), limit dextrinase (LD), the starch phosphorylator GWD and the glucan phosphatase dual-specificity phosphatase 4 (DSP4), also named starch excess 4 (SEX4), are reductively activated in vitro. Redox screens now suggest the presence of a substantially more extensive and coordinated redox regulation involving a larger number of enzymes. Noticeably several of these enzymes contain a new type of low-affinity carbohydrate-binding module that we term a low-affinity starch-binding domain or LA-SBD. These are present in the CBM20, CBM45 and CBM53 families and can enable diurnal dynamics of starch–enzyme recognition. Such diurnal changes in starch binding have been indicated for the redox-regulated GWD and SEX4.     

Genetic engineering of transitory starch accumulation by knockdown of OsSEX4 in rice plants for enhanced bioethanol production

Rice straw, a common agricultural waste, is used as a potential feedstock for bioethanol production. Currently, bioethanol is made mostly from the microbial fermentation of starch-containing raw materials. Therefore, genetically engineered starch-excess rice straw through interference of starch degradation as a potential strategy to enhance bioethanol production was evaluated in this study. Arabidopsis Starch Excess 4 (SEX4) encodes a chloroplast-localized glucan phosphatase and plays a role in transitory starch degradation. Despite the identification of a SEX4 homolog in rice, OsSEX4, its biological function remains uncertain. Ectopic expression of OsSEX4 complementary DNA complemented the leaf starch-excess phenotype of the Arabidopsis sex4-4 mutant. OsSEX4-knockdown transgenic rice plants were generated using the RNA interference approach. Starch accumulation was higher in OsSEX4-knockdown suspension-cultured cells, leaves, and rice straw compared with the wild type, suggesting that OsSEX4 plays an important role in degradation of transitory starch. The OsSEX4-knockdown rice plants showed normal plant growth and no yield penalty. Starch-excess OsSEX4-knockdown rice straw used as feedstock for fermentation resulted in improved bioethanol yield, with a 50% increase in ethanol production in a vertical mass-flow type bioreactor, compared with that of the wild-type straw.     

The Two Plastidial Starch-Related Dikinases Sequentially Phosphorylate Glucosyl Residues at the Surface of Both the A- and B-Type Allomorphs of Crystallized Maltodextrins But the Mode of Action Differs

In this study, two crystallized maltodextrins were generated that consist of the same oligoglucan pattern but differ strikingly in the physical order of double helices. As revealed by x-ray diffraction, they represent the highly ordered A- and B-type allomorphs. Both crystallized maltodextrins were similar in size distribution and birefringence. They were used as model substrates to study the consecutive action of the two starch-related dikinases, the glucan, water dikinase and the phosphoglucan, water dikinase. The glucan, water dikinase and the phosphoglucan, water dikinase selectively esterify glucosyl residues in the C6 and C3 positions, respectively. Recombinant glucan, water dikinase phosphorylated both allomorphs with similar rates and caused complete glucan solubilization. Soluble neutral maltodextrins inhibited the glucan, water dikinase-mediated phosphorylation of crystalline particles. Recombinant phosphoglucan, water dikinase phosphorylated both the A- and B-type allomorphs only following a prephosphorylation by the glucan, water dikinase, and the activity increased with the extent of prephosphorylation. The action of the phosphoglucan, water dikinase on the prephosphorylated A- and B-type allomorphs differed. When acting on the B-type allomorph, by far more phosphoglucans were solubilized as compared with the A type. However, with both allomorphs, the phosphoglucan, water dikinase formed significant amounts of monophosphorylated phosphoglucans. Thus, the enzyme is capable of acting on neutral maltodextrins. It is concluded that the actual carbohydrate substrate of the phosphoglucan, water dikinase is defined by physical rather than by chemical parameters. A model is proposed that explains, at the molecular level, the consecutive action of the two starch-related dikinases.In terms of quantity, starch is one of the most prominent photosynthesis-derived products. The global starch production by land plants has been estimated to be approximately 2,850 million tons per year (Burrell, 2003). Starch is highly relevant for nutrition in animals and humans, but it is also used for many industrial applications, such as additives in paper or textiles and in pharmacy products as well. In addition, starch appears to be increasingly important as a photosynthesis-based renewable energy source that can be converted into technologically relevant products such as bioethanol and hydrogen (Hannah and James, 2008; Zhang et al., 2008).Native starch is formed as a water-insoluble particle called a granule that is thought to comprise two types of polyglucans, amylopectin and amylose. The latter is an almost unbranched α-1,4-glucan and usually is the minor constituent of the starch particle, accounting for 10% to 35% of the total starch dry weight (Ball, 2000). However, in some mutants, the relative amylose content is strongly diminished, resulting in an essentially amylose-free starch (such as in the waxy mutant of maize [Zea mays]), or, alternatively, it is increased, forming up to 70% of the starch mass (e.g. in the amylose extender mutant from maize; Gérard et al., 2001). Nevertheless, in wild-type starches, amylopectin typically is the major constituent that also is essential for the molecular organization of the glucans within the entire starch granule (Ball and Morell, 2003). Like glycogen, amylopectin is a branched α-glucan with 4% to 6% of the inter-Glc linkages being α-1,6-bonds (Ball, 2000); however, as opposed to glycogen, the branching points occur as intramolecular clusters. Due to the length distribution of the side chains and the clustering of the branching points, neighboring glucan chains are capable of forming highly ordered double helices (Smith, 2001; Zeeman et al., 2002).As revealed by x-ray diffraction analysis, two major native starch structures are known that differ in the arrangement of the double helices. The A-type allomorph, which is typical of wild-type cereal starches but also occurs in lower plants, is more compact, as compared with the B type, and consists of flat layers of double helices. By contrast, in the B-type allomorph, six double helices are thought to surround a central cavity that is filled with water molecules. The B-type allomorph is found in starch synthesized by dicotyledonal storage organs, such as potato (Solanum tuberosum) tubers, in some high-amylose starches from cereal mutants (Gallant et al., 1997; Gérard et al., 2001), and in assimilatory starches from potato and Arabidopsis (Arabidopsis thaliana) as well (Hejazi et al., 2008). Legume starches are believed to represent another allomorph that is designated the C type. However, this allomorph is actually a mixture of both the A- and B-type crystallites within a single native starch particle rather than a third distinct type of the double helical arrangement (Imberty et al., 1991; Bogracheva et al., 2001).It should be noted that both the A- and B-type allomorphs of native starch granules often contain, as a minor constituent, an additional crystal structure designated the V type. Unlike the A- and B-type allomorphs, the V type is assumed to arise from single amylose helices, some of which are complexed with endogenous granular lipids. When estimated for the dry state, the V-type crystal structure accounts for only a small percentage of the total starch granule crystallinity (Lopez-Rubio et al., 2008).The physical structure of the native starch particle is likely to have important biochemical implications, as it affects the performance of carbohydrate-active enzymes and, thereby, the transition of carbohydrates from the solid phase to the soluble phase. This conclusion has been reached by in vitro experiments demonstrating that the pancreas α-amylase hydrolyzes A-type starch faster than the B-type counterpart (Gérard et al., 2001).Another metabolically important feature of amylopectin is the occurrence of covalent modification by phosphate esters that are found in a small proportion of the glucosyl residues. Most frequently phosphorylation occurs at the C6 position of the glucosyl residue, but C3 and, to a minor extent, C2 can also be esterified (Hizukuri et al., 1970). Recently, evidence has been presented that the esterification of the C6 and C3 positions of glucosyl residues differs in the structural effects on the neighboring inter-Glc bonds (Hansen et al., 2009). Phosphorylation at C6 is mediated by the recently identified α-glucan, water dikinase (GWD; EC 2.7.9.4), which utilizes ATP as dual phosphate donor and three distinct acceptors, two of which are sequentially used. The enzyme transfers the terminal phosphate group to water (thereby forming orthophosphate) and the β-phosphate group first to a conserved His residue within the catalytic domain of the monomeric GWD and, subsequently, to the C6 target of the glucosyl residue to be phosphorylated (Ritte et al., 2002, 2006). Phosphorylation at C3 is catalyzed by a second dikinase, designated phosphoglucan, water dikinase (PWD; EC 2.7.9.5; Ritte et al., 2006). The amino acid sequence of the catalytic (C-terminal) domain of PWD shares similarity with that of GWD, and in principle, the PWD-mediated catalysis follows the same mode of action as GWD, including the transient autophosphorylation at a conserved His residue (Baunsgaard et al., 2005; Kötting et al., 2005). However, PWD deviates from GWD in the amino acid sequence of the N-terminal domain, especially in the carbohydrate-binding region. PWD possesses a single carbohydrate-binding module that has been grouped into the family CBM20 (Machovič and Janaček, 2006a, 2006b). By contrast, the N-terminal domain of GWD contains two putative carbohydrate-binding motifs similar to those of an α-amylase that presumably is located in the chloroplasts (Yu et al., 2005). However, the structure of these motifs is still not known; therefore, a sequence-based prediction of the actual carbohydrate target is not yet possible.GWD- and PWD-deficient Arabidopsis mutants possess to some extent similar but not equal phenotypes. Leaves of GWD-deficient lines (which contain essentially unchanged levels of functional PWD) have starch levels that are at least five times higher than those of the wild type and remain high even after prolonged darkness. Growth of the entire plant is strongly compromised. The phenotype of PWD-deficient mutants (which express functional GWD) is less severe, as growth is only slightly diminished and transitory starch levels are elevated but not as strongly as in the GWD-deficient lines. Mutants lacking functional PWD can degrade transitory starch, but net degradation occurs at a lower rate as compared with wild-type plants (Kötting et al., 2005). These data clearly indicate that, in vivo, PWD cannot substitute for GWD and that glucosyl 6-phosphate residues are involved in a more strict control of the starch turnover as compared with the C3 phosphate esters.When considering the metabolic function(s) of starch phosphorylation, it should be noted that phosphorylation occurs during both net starch synthesis and degradation, although the rates of phosphorylation are likely to be different (Nielsen et al., 1994; Ritte et al., 2004). It is reasonable, therefore, to assume that starch phosphorylation exerts an important role in the entire transitory starch metabolism, rather than being functional only during the degrading process (and, consequently, the starch-related dikinases cannot, in a strict sense, be considered as “starch-degrading enzymes”).Depending on the botanical source, the degree of starch phosphorylation varies strongly. In potato tuber starch, approximately 0.1% to 0.5% of the glucosyl residues are phosphorylated (Ritte et al., 2002), and this value is considered to be indicative of a high level of phosphorylation. By contrast, cereal starches contain a far lower relative phosphate content that often is close to the limit of detection (approximately 0.002%; Glaring et al., 2006). In principle, these differences could be due to different rates of phosphorylation, as catalyzed by the two starch-related dikinases, and this assumption seems to be supported by the observation that, in general, starches of the B-type allomorph appear to have a higher degree of phosphorylation as compared with those of the A-type allomorph. If so, the dikinases may preferentially act on the B-type allomorph. Alternatively, the phosphorylation catalyzed by the two dikinases could be balanced by counteracting phosphatases, such as SEX4. This plastidial enzyme has been shown to act as a (phospho)glucan phosphatase that is involved in leaf starch metabolism (Kötting et al., 2009). If antagonistic enzyme activities are taken into consideration, the actual level of starch phosphorylation is determined by the rate of both phosphorylation and the subsequent hydrolysis of phosphate esters and, consequently, does not necessarily reflect the action of the starch-related dikinases.Recently, crystallized maltodextrins (MD_cryst) have been prepared that, by using x-ray diffraction, were identified as being the B-type allomorph and to possess a highly ordered structure (which exceeds that of native starch granules). MD_cryst have been applied as a substrate for a recombinant GWD from potato. Using a carefully optimized assay, the rate of phosphorylation was by far higher than that observed with any other carbohydrate substrate, such as native starch granules or starch-derived polysaccharides. By contrast, solubilization by heat treatment of the MD_cryst almost completely abolished the activity of GWD. Phosphorylation resulted in the formation of singly, doubly, and triply phosphorylated glucans and favored the solubilization of both neutral glucans and phosphoglucans (Hejazi et al., 2008). Recombinant PWD also phosphorylated MD_cryst, provided the MD_cryst had been prephosphorylated by GWD and were not solubilized by heat treatment (Hejazi et al., 2008).Because of the high phosphorylation rates and the phosphorylation pattern obtained, MD_cryst are a suitable model carbohydrate that mimics phosphorylation-relevant features of highly ordered regions within the native starch granule. It allows study of the action of the two starch-related dikinases and the transition of carbohydrates from the solid to the soluble state without any other starch-related enzyme being required.Until now, only the B-type allomorph of the MD_cryst has been applied as substrate of the two dikinases. Using native starch granules as a target, the rates of phosphorylation as obtained with recombinant GWD varied largely within the B-type allomorph (Hejazi et al., 2008); therefore, it is reasonable to assume that additional but largely unknown features of the native starch granule also strongly affect the action of GWD. This implies that any preference or specificity of the starch-related dikinases for a given allomorph can be analyzed most convincingly if MD_cryst preparations representing both the B- and A-type allomorphs are available.In this study, we used two MD_cryst preparations that are indistinguishable in their oligoglucan patterns but differ in the physical arrangement of the double helices and represent the highly ordered A- and B-type allomorphs. Using these two MD_cryst preparations, we analyzed the action of the two starch-related dikinases. The size distribution of the MD_cryst particles has been determined using the Coulter counter, and surface properties of both allomorphs were monitored by scanning electron microscopy. Thermal stability of the two allomorphs was analyzed by measuring the temperature dependence of light scattering. Finally, the phosphorylation-dependent solubilization of both allomorphs and the transition of (phospho)glucans into the soluble state have been studied.     

Light induces phosphorylation of glucan water dikinase, which precedes starch degradation in turions of the duckweed Spirodela polyrhiza

Degradation of storage starch in turions, survival organs of Spirodela polyrhiza, is induced by light. Starch granules isolated from irradiated (24 h red light) or dark-stored turions were used as an in vitro test system to study initial events of starch degradation. The starch-associated pool of glucan water dikinase (GWD) was investigated by two-dimensional gel electrophoresis and by western blotting using antibodies raised against GWD. Application of this technique allowed us to detect spots of GWD, which are light induced and absent on immunoblots prepared from dark-adapted plants. These spots, showing increased signal intensity following incubation of the starch granules with ATP, became labeled by randomized [betagamma-33P]ATP but not by [gamma-33P]ATP and were removed by acid phosphatase treatment. This strongly suggests that they represent a phosphorylated form(s) of GWD. The same light signal that induces starch degradation was thus demonstrated for the first time to induce autophosphorylation of starch-associated GWD. The in vitro assay system has been used to study further effects of the light signal that induces autophosphorylation of GWD and starch degradation. In comparison with starch granules from dark-adapted plants, those from irradiated plants showed increase in (1) binding capacity of GWD by ATP treatment decreased after phosphatase treatment; (2) incorporation of the beta-phosphate group of ATP into starch granules; and (3) rate of degradation of isolated granules by starch-associated proteins, further enhanced by phosphorylation of starch. The presented results provide evidence that autophosphorylation of GWD precedes the initiation of starch degradation under physiological conditions.     

The binding of α-amylase to starch plays a decisive role in the initiation of storage starch degradation in turions of Spirodela polyrhiza

Degradation of reserve starch in turions, perennation organs of the duckweed Spirodela polyrhiza , is induced by continuous red light (cR). Irradiation of the turions with this light results in the autophosphorylation of starch-associated glucan water dikinase (GWD). The ensuing phosphorylation of the starch by this enzyme was proposed to result in the enhanced association of starch-degrading enzymes to the starch granules and in the initiation of starch breakdown. The present results confirm that the irradiation of dark-adapted turions with cR results in phosphorylation of the starch, accompanying changes in the capacity of the granule starch to bind turion endogenous α-amylase, as well as changes in the starch degradation level. All three effects show very similar dependence on the time of irradiation, suggesting that they may be linked. The α-amylase is a plausible candidate for effecting starch breakdown initiation. However, the increased binding capacity of the starch granules for this enzyme is insufficient to account for the initiation of the starch breakdown as this capacity is already high prior to the irradiation. The decisive effect of cR irradiation on starch degradation may lie in enabling α-amylase to gain access to otherwise sequestered starch granules or in activating α-amylase bound to the granules.     

BIOCHEMICAL CYTOLOGY OF TRICHOMONAD FLAGELLATES : I. Subcellular Localization of Hydrolases, Dehydrogenases, and Catalase in Tritrichomonas foetus

To determine the localization of several enzymes in Tritrichomonas foetus, the axenic KV-1 strain was grown in Diamond's medium with bovine serum, homogenized in 0.25 M sucrose, and subjected to analytical differential and isopycnic centrifugation. The fractions were assayed for their enzymatic composition and examined electron microscopically. NADH and NADPH dehydrogenases, about 90% of the catalase, and two hydrolases, α-galactosidase and manganese-activated β-galactosidase I are in the nonsedimentable part of the cytoplasm. α-Glycerophosphate and malate dehydrogenases are associated with a large particle, whose equilibrium density in sucrose gradients is 1.24. This particle corresponds to that population of the paracostal and paraxostylar granules which, having a uniform granular matrix surrounded by a single membrane, resemble microbodies from other organisms. The small sedimentable portion of catalase (about 10% of the total activity) is not associated with these granules and equilibrates at density 1.22. The nature of the subcellular entity carrying catalase could not be ascertained. Hydrolases with a pH optimum around 6–6.5 (protease, β-N-acetylglucosaminidase, β-N-acetylgalactosaminidase, and cation-independent β-galactosidase II), as well as a large part of acid phosphatase, are associated with a population of large particles which equilibrate at densities from 1.15 to 1.20. The hydrolases in these granules lose their structure-bound latency easily after freezing and thawing. These particles correspond to another population of the paracostal and paraxostylar granules which have varied shape and inhomogeneous content with frequent myelin figures, indicating a digestive function. The rest of the phosphatase and most of the acid β-glucuronidase activity are in a smaller granule fraction with an equilibrium density around 1.18. The latency of these enzymes is quite resistant to freezing and thawing. This particle population consists of smaller, very often flattened vesicles and granules, many of which are clearly fragments of the prominent Golgi apparatus of the cell.     

The Simultaneous Abolition of Three Starch Hydrolases Blocks Transient Starch Breakdown in Arabidopsis

In this study, we investigated which enzymes are involved in debranching amylopectin during transient starch degradation. Previous studies identified two debranching enzymes, isoamylase 3 (ISA3) and limit dextrinase (LDA), involved in this process. However, plants lacking both enzymes still degrade substantial amounts of starch. Thus, other enzymes/mechanisms must contribute to starch breakdown. We show that the chloroplastic α-amylase 3 (AMY3) also participates in starch degradation and provide evidence that all three enzymes can act directly at the starch granule surface. The isa3 mutant has a starch excess phenotype, reflecting impaired starch breakdown. In contrast, removal of AMY3, LDA, or both enzymes together has no impact on starch degradation. However, removal of AMY3 or LDA in addition to ISA3 enhances the starch excess phenotype. In plants lacking all three enzymes, starch breakdown is effectively blocked, and starch accumulates to the highest levels observed so far. This provides indirect evidence that the heteromultimeric debranching enzyme ISA1-ISA2 is not involved in starch breakdown. However, we illustrate that ISA1-ISA2 can hydrolyze small soluble branched glucans that accumulate when ISA3 and LDA are missing, albeit at a slow rate. Starch accumulation in the mutants correlates inversely with plant growth.     

Systematic Analysis of Pericarp Starch Accumulation and Degradation during Wheat Caryopsis Development

Although wheat (Triticum aestivum L.) pericarp starch granule (PSG) has been well-studied, our knowledge of its features and mechanism of accumulation and degradation during pericarp growth is poor. In the present study, developing wheat caryopses were collected and starch granules were extracted from their pericarp to investigate the morphological and structural characteristics of PSGs using microscopy, X-ray diffraction and Fourier transform infrared spectroscopy techniques. Relative gene expression levels of ADP-glucose pyrophosphorylase (APGase), granule-bound starch synthase II (GBSS II), and α-amylase (AMY) were quantified by quantitative real-time polymerase chain reaction. PSGs presented as single or multiple starch granules and were synthesized both in the amyloplast and chloroplast in the pericarp. PSG degradation occurred in the mesocarp, beginning at 6 days after anthesis. Amylose contents in PSGs were lower and relative degrees of crystallinity were higher at later stages of development than at earlier stages. Short-range ordered structures in the external regions of PSGs showed no differences in the developing pericarp. When hydrolyzed by α-amylase, PSGs at various developmental stages showed high degrees of enzymolysis. Expression levels of AGPase, GBSS II, and AMY were closely related to starch synthesis and degradation. These results help elucidate the mechanisms of accumulation and degradation as well as the functions of PSG during wheat caryopsis development.     

Enzymes of starch metabolism in the developing rice grain

The levels of starch, soluble sugars, protein, and enzymes involved in starch metabolism—α-amylase, β-amylase, phosphorylase, Q-enzyme, R-enzyme, and starch synthetase —were assayed in dehulled developing rice grains (Oryzasativa L., variety IR8). Phosphorylase, Q-enzyme, and R-enzyme had peak activities 10 days after flowering, whereas α- and β-amylases had maximal activities 14 days after flowering. Starch synthetase bound to the starch granule increased in activity up to 21 days after flowering. These enzymes (except the starch synthetases) were also detected by polyacrylamide gel electrophoresis. Their activity in grains at the midmilky stage (8-10 days after flowering) was determined in five pairs of lines with low and high amylose content from different crosses. The samples had similar levels of amylases, phosphorylase, R-enzyme, and Q-enzyme. The samples consistently differed in their levels of starch synthetase bound to the starch granule, which was proportional to amylose content. Granule-bound starch synthetase may be responsible for the integrity of amylose in the developing starch granule.     

Identification of a novel enzyme required for starch metabolism in Arabidopsis leaves. The phosphoglucan, water dikinase

The phosphorylation of amylopectin by the glucan, water dikinase (GWD; EC 2.7.9.4) is an essential step within starch metabolism. This is indicated by the starch excess phenotype of GWD-deficient plants, such as the sex1-3 mutant of Arabidopsis (Arabidopsis thaliana). To identify starch-related enzymes that rely on glucan-bound phosphate, we studied the binding of proteins extracted from Arabidopsis wild-type leaves to either phosphorylated or nonphosphorylated starch granules. Granules prepared from the sex1-3 mutant were prephosphorylated in vitro using recombinant potato (Solanum tuberosum) GWD. As a control, the unmodified, phosphate free granules were used. An as-yet uncharacterized protein was identified that preferentially binds to the phosphorylated starch. The C-terminal part of this protein exhibits similarity to that of GWD. The novel protein phosphorylates starch granules, but only following prephosphorylation with GWD. The enzyme transfers the beta-P of ATP to the phosphoglucan, whereas the gamma-P is released as orthophosphate. Therefore, the novel protein is designated as phosphoglucan, water dikinase (PWD). Unlike GWD that phosphorylates preferentially the C6 position of the glucose units, PWD phosphorylates predominantly (or exclusively) the C3 position. Western-blot analysis of protoplast and chloroplast fractions from Arabidopsis leaves reveals a plastidic location of PWD. Binding of PWD to starch granules strongly increases during net starch breakdown. Transgenic Arabidopsis plants in which the expression of PWD was reduced by either RNAi or a T-DNA insertion exhibit a starch excess phenotype. Thus, in Arabidopsis leaves starch turnover requires a close collaboration of PWD and GWD.