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Information on EC 2.7.9.4 - alpha-glucan, water dikinase and Organism(s) Arabidopsis thaliana and UniProt Accession Q9SAC6
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2.7.9.4 alpha-glucan, water dikinaseIUBMB Comments
Requires Mg2+. ATP appears to be the only phosphate donor. No activity could be detected using GTP, UTP, phosphoenolpyruvate or diphosphate . The protein phosphorylates glucans exclusively on O-6 of glucosyl residues . The protein phosphorylates itself with the beta-phosphate of ATP, which is then transferred to the glucan .
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Word Map
- 2.7.9.4
- glucans
- ribonucleotide
- deoxyribonucleotides
- alpha-glucans
- phosphoglucans
- amylopectin
- beta-amylase
-
allomorph
-
spirodela
-
beta-phosphate
- turions
-
starch-binding
- polyrhiza
- food industry
- biotechnology
The taxonomic range for the selected organisms is: Arabidopsis thaliana
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria
Synonyms
r1 protein, glucan water dikinase, alpha-glucan water dikinase, atgwd3, atgwd2, starch-related r1 protein, glucan water dikinase 2, glucan water dikinase 3, starch-related alpha-glucan/water dikinase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
At1g10760
alpha-glucan, water dikinase, GWD
-
-
-
-
At4g24450
AtGWD2
AtGWD3
GWD
starch-related R1 protein
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
methyl group transfer
-
-
-
-
PATHWAY SOURCE
PATHWAYS
-
-
SYSTEMATIC NAME
IUBMB Comments
ATP:alpha-glucan, water phosphotransferase
Requires Mg2+. ATP appears to be the only phosphate donor. No activity could be detected using GTP, UTP, phosphoenolpyruvate or diphosphate [1]. The protein phosphorylates glucans exclusively on O-6 of glucosyl residues [2]. The protein phosphorylates itself with the beta-phosphate of ATP, which is then transferred to the glucan [1].
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ATP + crystalline maltodextrin + H2O
AMP + phospho-alpha-glucosyl-maltodextrin + phosphate
crystalline maltodextrin (MDcryst) is used as a model substrate for glucan phosphorylating enzyme activity that mimics features of native starches, such as allomorph and crystallinity but omitted branching. MDcryst has a higher degree of crystallinity and, therefore, recombinant GWD phosphorylates MDcryst with much higher rates than any other native starches tested so far. The incorporation of phosphate esters results in the release of phosphorylated (single, double, and triple phosphorylated glucan chains) as well as neutral maltodextrins from the water-insoluble MDcryst, indicating that the action of GWD disrupts the ordered arrangement of maltodextrins at the particle surface
-
-
?
ATP + amylopectin + H2O
?
-
phosphorylation at the C3 position of glucose residues in amylopectin
-
-
?
ATP + amylopectin + H2O
AMP + phospho-amylopectin + phosphate
1 h, 30°C, pH 7, in presence of 10 mM NH4Cl, 10 mM MgCl2, 0.5 mM dithiothreitol, 0.2 mg/ml bovine serum albumin, substrates: waxy maize amylopectin (unmodified (glycogen), elongated by phosphorylase a, pre-phosphorylated by potato glucan, water dikinase (GWD), or elongated and pre-phosphorylated) or soluble potato starch
reaction stop by boiling
-
?
ATP + crystalline maltodextrin + H2O
AMP + phospho-alpha-glucosyl-maltodextrin + phosphate
crystalline maltodextrin (MDcryst) is used as a model substrate for glucan phosphorylating enzyme activity that mimics features of native starches, such as allomorph and crystallinity but omitted branching. MDcryst has a higher degree of crystallinity and, therefore, recombinant GWD phosphorylates MDcryst with much higher rates than any other native starches tested so far. The incorporation of phosphate esters results in the release of phosphorylated (single, double, and triple phosphorylated glucan chains) as well as neutral maltodextrins from the water-insoluble MDcryst, indicating that the action of GWD disrupts the ordered arrangement of maltodextrins at the particle surface
-
-
?
ATP + crystalline maltodextrin + H2O
AMP + phosphorylated crystalline maltodextrin + phosphate
-
-
-
-
?
ATP + starch + H2O
AMP + phosphorylated starch + phosphate
-
-
-
?
ATP + alpha-glucan + H2O
AMP + phospho-alpha-glucan + phosphate
-
-
-
?
ATP + alpha-glucan + H2O
AMP + phospho-alpha-glucan + phosphate
dikinases use ATP as a dual phosphate donor and transfer the beta- and gamma-phosphate groups to two distinct acceptor molecules, a glucan and water. A conserved histidine residue within this domain is capable of accepting the beta-phosphate group of ATP followingnucleotide binding and hydrolysis
-
-
?
ATP + alpha-glucan + H2O
AMP + phospho-alpha-glucan + phosphate
GWD phosphorylates the hydroxyl group at carbon atoms 6
-
-
?
ATP + alpha-glucan + H2O
AMP + phospho-alpha-glucan + phosphate
GWD phosphorylates the hydroxyl group at carbon atoms 6 and 3, the gamma-phosphate group of ATP is transferred to water and the beta-phosphate group to an autocatalytical histidine residue via a phosphoramidate bond. The phosphoramidate bond is acid labile, but rather stable under alkaline conditions. The gamma-phosphate group is transferred to water. GWD-dependent incorporation of starch phosphate monoesters is higher after removing these surface-exposed glucans by amylolytic enzymes
-
-
?
amylopectin + ATP + H2O
AMP + phospho-amylopectin + phosphate
-
essential step within starch metabolism. In Arabidopsis leaves starch turnover requires a close collaboration of alpha-glucan, water dikinase and phosphoglucan, water dikinase
-
-
?
amylopectin + ATP + H2O
AMP + phospho-amylopectin + phosphate
-
the endophytic fungus Piriformospora indica stimulates the expression of glucan-water dikinase Arabidopsis roots through a homeodomain transcription factor that binds to a conserved motif in the promoter
-
-
?
amylopectin + ATP + H2O
AMP + phospho-amylopectin + phosphate
-
the enzyme is involved in the cold-induced development of freezing tolerance in Arabidopsis
-
-
?
ATP + alpha-glucan + H2O
AMP + phospho-alpha-glucan + phosphate
-
-
-
-
?
ATP + alpha-glucan + H2O
AMP + phospho-alpha-glucan + phosphate
exclusive phosphorylation of C-6 position, in vitro
-
-
?
ATP + alpha-glucan + H2O
AMP + phospho-alpha-glucan + phosphate
-
phosphorylation at C-6 position, crystallized maltodextrin, natural starch from maize, potato, Arabidopsis, A- and B-allomorph type
-
-
?
ATP + alpha-glucan + H2O
AMP + phospho-alpha-glucan + phosphate
dikinases use ATP as a dual phosphate donor and transfer the beta- and gamma-phosphate groups to two distinct acceptor molecules, a glucan and water
-
-
?
ATP + alpha-glucan + H2O
AMP + phospho-alpha-glucan + phosphate
GWD phosphorylates the hydroxyl group at carbon atom 6, the gamma-phosphate group of ATP is transferred to water and the beta-phosphate group to an autocatalytical histidine residue via a phosphoramidate bond
-
-
?
phospho-amylopectin + ATP + H2O
AMP + phospho-amylopectin + phosphate
-
the enzyme is involved in degradation of transistory starch
-
-
?
phospho-amylopectin + ATP + H2O
AMP + phospho-amylopectin + phosphate
-
AtGWD3 primarily catalyzes phosphorylation at the C-3 position of the glucose unit of preferably pre-phosphorylated amylopectin substrate with lon side chains
-
-
?
additional information
?
-
the activity of GWD correlates with the amount of accessible glucan chains on the starch granule surface
-
-
?
additional information
?
-
-
the activity of GWD correlates with the amount of accessible glucan chains on the starch granule surface
-
-
?
additional information
?
-
no phosphorylation of the hydroxyl group at the C2 position
-
-
?
additional information
?
-
no phosphorylation of the hydroxyl group at the C2 position
-
-
?
additional information
?
-
-
alpha-glucan phosphorylation enhances beta-amylolytic starch degradation and vice versa: effective starch mobilization dependent on simultaneous action of GDW and beta-amylase
-
-
?
additional information
?
-
not involved in transient starch degradation, plants lacking AtGWD show similar starch and sugar content as wild-type plants
-
-
?
additional information
?
-
-
not involved in transient starch degradation, plants lacking AtGWD show similar starch and sugar content as wild-type plants
-
-
?
additional information
?
-
-
phosphorylation by GWD mediates phase transition from highly ordered to hydrated state glucans
-
-
?
additional information
?
-
-
phosphorylation of crystalline maltodextrin initiates solubilisation of both phosphorylated and neutral glucans
-
-
?
additional information
?
-
-
substrate-specificity defined by physical arrangements of alpha glucans and by molecular order of glucan helices
-
-
?
additional information
?
-
activity is independent of pre-phosphorylation of glucan substrate
-
-
?
additional information
?
-
-
activity is independent of pre-phosphorylation of glucan substrate
-
-
?
additional information
?
-
-
crystallized maltodextrin represents B-type starch allomorph, 30 min or 1 h, linear phosphorylation kinetics with respect to time and enzyme concentration (up to 1 h), phosphorylation at the surface of the crystalline maltodextrin, formation of a wide range of phosphoglucans (detection of mono-, di, and triphosphorylated alpha glucans), initiation of solubilisation of both phosphoglucans and neutral glucans from the crystalline maltodextrin
-
-
?
additional information
?
-
-
GDW activity stimulates release of maltose from granular starch (by beta-amylase activity)
-
-
?
additional information
?
-
-
GWD3-SBD shows strongest binding towards alpha-cyclodextrin and no detectable affinity for linear maltooligosaccharides or for phosphorylated beta-cyclodextrin
-
-
?
additional information
?
-
no phosphorylation of the hydroxyl group at the C2 position
-
-
?
additional information
?
-
no phosphorylation of the hydroxyl group at the C2 position
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ATP + amylopectin + H2O
?
-
phosphorylation at the C3 position of glucose residues in amylopectin
-
-
?
phospho-amylopectin + ATP + H2O
AMP + phospho-amylopectin + phosphate
-
the enzyme is involved in degradation of transistory starch
-
-
?
ATP + alpha-glucan + H2O
AMP + phospho-alpha-glucan + phosphate
-
-
-
?
ATP + alpha-glucan + H2O
AMP + phospho-alpha-glucan + phosphate
dikinases use ATP as a dual phosphate donor and transfer the beta- and gamma-phosphate groups to two distinct acceptor molecules, a glucan and water. A conserved histidine residue within this domain is capable of accepting the beta-phosphate group of ATP followingnucleotide binding and hydrolysis
-
-
?
ATP + alpha-glucan + H2O
AMP + phospho-alpha-glucan + phosphate
GWD phosphorylates the hydroxyl group at carbon atoms 6
-
-
?
amylopectin + ATP + H2O
AMP + phospho-amylopectin + phosphate
-
essential step within starch metabolism. In Arabidopsis leaves starch turnover requires a close collaboration of alpha-glucan, water dikinase and phosphoglucan, water dikinase
-
-
?
amylopectin + ATP + H2O
AMP + phospho-amylopectin + phosphate
-
the endophytic fungus Piriformospora indica stimulates the expression of glucan-water dikinase Arabidopsis roots through a homeodomain transcription factor that binds to a conserved motif in the promoter
-
-
?
amylopectin + ATP + H2O
AMP + phospho-amylopectin + phosphate
-
the enzyme is involved in the cold-induced development of freezing tolerance in Arabidopsis
-
-
?
ATP + alpha-glucan + H2O
AMP + phospho-alpha-glucan + phosphate
exclusive phosphorylation of C-6 position, in vitro
-
-
?
ATP + alpha-glucan + H2O
AMP + phospho-alpha-glucan + phosphate
dikinases use ATP as a dual phosphate donor and transfer the beta- and gamma-phosphate groups to two distinct acceptor molecules, a glucan and water
-
-
?
additional information
?
-
the activity of GWD correlates with the amount of accessible glucan chains on the starch granule surface
-
-
?
additional information
?
-
-
the activity of GWD correlates with the amount of accessible glucan chains on the starch granule surface
-
-
?
additional information
?
-
-
alpha-glucan phosphorylation enhances beta-amylolytic starch degradation and vice versa: effective starch mobilization dependent on simultaneous action of GDW and beta-amylase
-
-
?
additional information
?
-
not involved in transient starch degradation, plants lacking AtGWD show similar starch and sugar content as wild-type plants
-
-
?
additional information
?
-
-
not involved in transient starch degradation, plants lacking AtGWD show similar starch and sugar content as wild-type plants
-
-
?
additional information
?
-
-
phosphorylation by GWD mediates phase transition from highly ordered to hydrated state glucans
-
-
?
additional information
?
-
-
phosphorylation of crystalline maltodextrin initiates solubilisation of both phosphorylated and neutral glucans
-
-
?
additional information
?
-
-
substrate-specificity defined by physical arrangements of alpha glucans and by molecular order of glucan helices
-
-
?
additional information
?
-
-
GWD3-SBD shows strongest binding towards alpha-cyclodextrin and no detectable affinity for linear maltooligosaccharides or for phosphorylated beta-cyclodextrin
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.02
amylopectin, mean chain length: 23.5, nmol glucose-6-phosphate/mg: 0.1
0.04
amylopectin, pre-phosphorylated, mean chain length: 24.8, nmol glucose-6-phosphate/mg: 0.5
0.49
amylopectin, elongated, mean chain length: 29.7, nmol glucose-6-phosphate/mg: 0.1
0.65
amylopectin, elongated and pre-phosphorylated, mean chain length: 30.8, nmol glucose-6-phosphate/mg: 39.4
additional information
-
conversion of heat solubilized maltodextrin below 1 nmol/min/mg
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
floral organ, strong expression in vascular tissue just before onset of senescence (promoter activity)
phloem, high expression just before onset of senescence (promoter activity)
strong expression in vascular tissue just before onset of senescence (promoter activity)
strong expression in vascular tissue just before onset of senescence (promoter activity)
leaf, stem, root, flower, silique only just before onset of senescence (promoter activity)
additional information
strong expression in vascular tissue (abaxial side of primary vascular bundle, phloem) just before onset of senescence (promoter activity), senescent leaves (microarray)
strong expression in vascular tissue just before onset of senescence (promoter activity)
strong expression in vascular tissue (floral organ abscission zone, dehiscence zone between silique walls, vascular tissue of silique walls) just before onset of senescence (promoter activity, microarray)
strong expression in vascular tissue just before onset of senescence (promoter activity)
additional information
-
enzyme is expressed in all starch-synthesizing tissues
additional information
spatially (vascular) and temporarily (age-dependent) restricted expression pattern (promoter activity)
additional information
-
spatially (vascular) and temporarily (age-dependent) restricted expression pattern (promoter activity)
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
along the cell wall, N-terminal 158 and 61 amino acids (revealed by confocal laser scanning microscopy)
the expression of AtGWD2 is in a relatively late stage of plant development
N-terminal 158 and 61 amino acids (revealed by confocal laser scanning microscopy)
additional information
altered GWD expression levels influence the surface properties of starch granules, determination of granule morphologies in different transgenic lines, which affects the starch synthesis by enzyme AtSS1, overview
-
the enzyme acts significantly on the surface of native starch granules
-
additional information
the GWD full-length protein binds to native starch granules in vivo and in vitro. Binding of GWD in vivo is dependent on the metabolic status of the cells. A significantly higher proportion of the dikinases is associated with native leaf starch granules isolated during the dark period than in the light phase of the photoperiod
-
additional information
the GWD full-length protein binds to native starch granules in vivo and in vitro. Binding of GWD in vivo is dependent on the metabolic status of the cells. A significantly higher proportion of the dikinases is associated with native leaf starch granules isolated during the dark period than in the light phase of the photoperiod
-
additional information
extra-plastidial localisation of full-length enzyme (in vitro chloroplast import assay), lack of chloroplast transit peptide
-
additional information
-
extra-plastidial localisation of full-length enzyme (in vitro chloroplast import assay), lack of chloroplast transit peptide
-
additional information
no fluorescence signal detected for full-length enzyme or its 610 N-terminal residues fused to eYFP or eGFP
-
additional information
-
no fluorescence signal detected for full-length enzyme or its 610 N-terminal residues fused to eYFP or eGFP
-
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
the largest differences in the amino acid sequence of GWD, EC 2.7.9.4, and PWD, EC 2.7.9.5, span the non-catalytic N-terminal region. In case of PWD, the N-terminus contains a single starch-binding domain (SBD) that belongs to the well-characterized carbohydrate-binding module (CBM) family CBM20. In contrast to PWD, the identity of the N-terminal starch-binding domain of GWD is less pronounced but might be assigned to the recently identified CBM45 family
malfunction
metabolism
physiological function
evolution
malfunction
metabolism
physiological function
malfunction
analysis of changes in GWD protein abundance in relation to starch levels in wild-type plants, in transgenic plants in which GWD transcripts are strongly reduced by induction of RNA interference, and in transgenic plants overexpressing GWD, overview. Overexpression of GWD does not accelerate starch degradation in leaves, and starch degradation is not inhibited until GWD levels are reduced by 70%. GWD protein levels do not vary over the diel cycle and the protein has a half-life of 2 days. Plants expressing redox-insensitive GWD have normal starch turnover
malfunction
GWD-deficient Arabidopsis thaliana mutants have a starch excess phenotype (sex1). The internal structure of granules isolated from GWD mutant plants, having reduced or no GWD activity, is unaffected, as thermal stability, allomorph, chain length distribution and density of starch granules are similar to wild-type, but short glucan chain residues located at the granule surface dominate in starches of transgenic plants and impede GWD activity. A similarly reduced rate of phosphorylation by GWD is also observed in potato tuber starch fractions that differ in the proportion of accessible glucan chain residues at the granule surface. A model is proposed to explain the characteristic morphology of starch granules observed in GWD transgenic plants. The model postulates that the occupancy rate of single glucan chains at the granule surface limits accessibility to starch-related enzymes
malfunction
mutants lacking the enzyme reveal a starch excess phenotype as well as growth retardation. The lack of GWD causes a reduction of G6P and G3P. The lack of GWD in mature Arabidopsis thaliana plants as observed in the null mutant sex1-8 results in a fivefold increased leaf starch content compared to wild-type plants. Although the internal structure of sex1-8 starch granules is similar to wild-type, the length as well as the abundance of glucan chains exposed on the granule surface differs in the mutant. The lack of GWD in mature sex1-8 plants results in a shift of the chain length distribution towards shorter glucan chains at the granule surface, the abundance of glucan chains at the granule surface is increased in the mutant compared to wild-type. Similar to the Arabidopsis GWD null mutant, the altered surface properties are also present in two partially complemented sex1-8 mutants which have compared to WT an approx. 79% and 93% lowered GWD protein level, respectively. Reduced expression of GWD impair the elongation of existing glucan chains catalyzed by starch synthase 1 (AtSS1), one of the dominate starch synthesizing activity in Arabidopsis. Impaired starch degradation caused by GWD deficiency may lead to lowered export of sugars to heterotrophic tissue and an overall reduction of biomass
metabolism
during starch metabolism, the phosphorylation of glucosyl residues of amylopectin is a repeatedly observed process. The phosphorylation is mediated by dikinases, the glucan, water dikinase (GWD, EC 2.7.9.4) and the phosphoglucan, water dikinase (PWD, EC 2.7.9.5). By the collaborative action of both enzymes, the initiation of a transition of alpha-glucans from highly ordered, water-insoluble state to a less order state is realized and thus the initial process of starch degradation
metabolism
glucan, water dikinase is a key enzyme of starch metabolism
metabolism
the first step on the pathway of starch degradation in Arabidopsis thaliana leaves at night is the phosphorylation of starch polymers, catalyzed by glucan, water dikinase, GWD. The enzyme exerts only a low level of control over starch degradation in Arabidopsis leaves. The starch-phosphorylating enzymes are attractive candidates for the control of flux through starch degradation. Daily control of starch degradation is likely to be at a posttranslational level
physiological function
the enzyme is involved in starch degradation
physiological function
enzyme GWD is a subject to redox regulation. A disulfide bond can be reduced in vitro by micromolar concentrations of reduced thioredoxins, resulting in activation of the enzyme. Enzyme GWD in the soluble fraction of plant extracts is in the reduced form, but a fraction of the enzyme bound to starch granules is reported to be in an oxidized, inactive form. Enzyme GWD has a long half-life and a low flux control coefficient for starch degradation. Transcriptional regulation of GWD protein levels is unlikely to be important for the control of starch degradation
physiological function
glucan phosphorylation mediated by alpha-glucan, water dikinase is essential in the light phase for a functional transitory starch turnover. The length of the light phase affects the phosphorylation state of the transitory starch and, by this, the average leaf starch content and the resulting growth of the plants. The longer the light period, the higher the starch phosphorylation rate at G-6 of alpha-glucan and the lower the starch content. Starch phosphorylation by GWD in the light phase is in parts necessary for proper starch degradation in the following night
physiological function
phosphorylation of transitory starch by alpha-glucan, water dikinase during starch turnover affects the surface properties and morphology of starch granules
physiological function
the starch-related dikinase utilizes ATP as dual phosphate donor transferring the terminal gamma-phosphate group to water selectively to C6 position of a glucosyl residue within amylopectin. The action of the dikinase is restricted to the granule surface and glucan chains exposed at the surface account only for a minor proportion of the entire granule. Glucan chains that are phosphorylated by the dikinase remain covalently linked to the insoluble starch particle. In Arabidopsis leaf starch about 0.1% of the glucosyl residues are phosphorylated, respectively. GWD is responsible for C3 and C6 phosphorylation. A significant PWD-mediated C3 phosphorylation requires the preceding phosphorylation by GWD in Arabidopsis thaliana wild-type starch. GWD preferentially acts on crystalline surfaces and GWD-mediated phosphorylation enables a phase transition at the granule surface from a solid to a more soluble state enabling a significant amylolysis
physiological function
important enzyme of starch metabolism. Catalyzes the addition of phosphate groups to amylopectin chains at the surface of starch granules, changing its physicochemical properties
evolution
the largest differences in the amino acid sequence of GWD, EC 2.7.9.4, and PWD, EC 2.7.9.5, span the non-catalytic N-terminal region. In case of PWD, the N-terminus contains a single starch-binding domain (SBD) that belongs to the well-characterized carbohydrate-binding module (CBM) family CBM20. In contrast to PWD, the identity of the N-terminal starch-binding domain of GWD is less pronounced but might be assigned to the recently identified CBM45 family
evolution
significant diversity in the evolution of alpha-glucan, water dikinase enzymes across plant species may be evolutionarily advantageous according to the varying needs for phosphorylated stored starch between plants and environments. Computational approaches to compare the enzyme sequences of 48 plant species provide an insight into the evolutionary variation in catalytic activity of alpha-glucan, water dikinase among plants. Deleterious mutations are identified for some plants at various positions of the five aromatic amino acids, which are highly conserved in tandems of CBM45 and vital for binding of the enzymes to starch. These mutations may be responsible for altered carbohydrate binding activity of alpha-glucan, water dikinase in plants, thereby affecting phosphorylation of transit and stored starch
malfunction
AtGWD2 knockout mutants do not accumulate high amounts of starch nor have a visible growth phenotype compared to wild-type plants
malfunction
gwd2 mutants are shrunken, with the epidermal cells of the seed coat irregularly shaped. gwd2 seeds contain a lower lipid to protein ratio and are impaired in germination
metabolism
during starch metabolism, the phosphorylation of glucosyl residues of amylopectin is a repeatedly observed process. The phosphorylation is mediated by dikinases, the glucan, water dikinase (GWD, EC 2.7.9.4) and the phosphoglucan, water dikinase (PWD, EC 2.7.9.5). By the collaborative action of both enzymes, the initiation of a transition of alpha-glucans from highly ordered, water-insoluble state to a less order state is realized and thus the initial process of starch degradation
metabolism
the enzyme catalyzes starch phosphorylation, an integral step in transitory starch degradation
physiological function
the starch-related dikinase utilizes ATP as dual phosphate donor transferring the terminal gamma-phosphate group to water selectively to C6 position of a glucosyl residue within amylopectin. The action of the dikinase is restricted to the granule surface and glucan chains exposed at the surface account only for a minor proportion of the entire granule. Glucan chains that are phosphorylated by the dikinase remain covalently linked to the insoluble starch particle. In Arabidopsis, leaf starch and potato tuber starch about 0.1 and 1% of the glucosyl residues are phosphorylated, respectively. A direct access of AtGWD2 to leaf starch granules in vivo and an overall impact on transitory starch metabolism is excluded
physiological function
the enzyme plays no role in transient starch metabolism. The enzyme (GWD2) possibly in concert with BAM5, may thus contribute to controlling the ratio between soluble and insoluble polysaccharides in plants
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). GWD1_ARATH
1399
0
156582
Swiss-Prot
Chloroplast (Reliability: 5)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
14200
-
theoretical, starch-binding domain, SBD, Asp68-Gly184, verified by SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
phosphoprotein
phosphoprotein
-
the protein possesses two vicinal phosphorylation sites, both of which are transiently phosphorylated
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C1019S
site-directed mutagenesis, the mutant is redox-insensitiv in contrast to the wild-type enzyme
H1004A
-
lacks autophosphorylation, fails to stimulate beta-amylase 3-catalyzed starch breakdown, similar binding to starch granules as wild-type enzyme (in vitro), H1004: conserved residue within phosphohistidine domain
additional information
additional information
construction of transgenic Arabidopsis thaliana plants, i.e. gwd-c1, gwd-c2, and gwd-c1/gwd-c2 or sex1-8, with reduced or no GWD activity. Absence of GWD results in altered starch granule morphology but no change in the physicochemical properties of starch granules, phenotype, overview. Partially complemented GWD transgenic lines exhibit an intermediate phenotype. Effects of the mutations on starch-related enzymes in the four Arabidopsis lines, overview
additional information
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construction of transgenic Arabidopsis thaliana plants, i.e. gwd-c1, gwd-c2, and gwd-c1/gwd-c2 or sex1-8, with reduced or no GWD activity. Absence of GWD results in altered starch granule morphology but no change in the physicochemical properties of starch granules, phenotype, overview. Partially complemented GWD transgenic lines exhibit an intermediate phenotype. Effects of the mutations on starch-related enzymes in the four Arabidopsis lines, overview
additional information
generation of transgenic plants expressing constitutively active GWD and of Arabidopsis plants expressing a dexamethasone-inducible silencing (RNAi) construct targeted at GWD. These plants retain normal control of starch degradation in leaves. Analysis of changes in GWD protein abundance in relation to starch levels in wild-type plants, in transgenic plants in which GWD transcripts are strongly reduced by induction of RNA interference, and in transgenic plants overexpressing GWD, overview
additional information
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in enzyme deficient mutant plants, phosphorylation at both C6- and C3-positions of glucose moieties in starch is dramatically decreased. In mutant plants lacking phosphoglucan, water dikinase EC 2.7.9.5, C3-bound phosphate is reduced to levels close to detection limit
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
by metal-affinity chromatography after expression in Saccharomyces cerevisiae with C-terminal V5- and hexa-His-tag
by metal-affinity chromatography on a His-Trap-HP column (elution: imidazole gradient, 50-500 mM), followed by buffer exchange and concentration
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recombinant GWD3-SBD is purified on a beta-cyclodextrin Sepharose column and by an additional His-trap purification step
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene gwd, recombinant expression of Arabidopsis thaliana GWD mutant C1019S C-terminally fused to GFP from a 35S promoter in transgenic Arabidopsis thaliana plants
gene GWD1, construction of transgenic plants expressing the soluble StrepII-tagged mutant enzyme, transformation via Agrobacterium tumefaciens mediation method
gene GWD1, the gene is located on chromosome 1, phylogenetic tree
heterologously expressed in potato tubers (Solanum tuberosum). Expression of AtGWD2 and AtGWD3 are not suitable for engineering starch with a high phosphate content in potato tubers but can provide new insights in the process of starch phosphorylation in the amyloplast
in pKGWFS7 for expression N-terminally of an (eGFP)-beta-glucoronidase fusion and Agrobacterium-mediated transformation of Arabidopsis plants, in pK7FWG2 (as full-length or 158 N-terminal amino acid fragment) for expression as eGFP-fusion and subcellular localization in onion epidermis and Arabidopsis thaliana, in pPS48uYFP as full-length, C-terminal truncations (N-terminal 610, 158, or 61 amino acids) or C-terminally fused to transit peptide of AtGWD isoform 1 for Agrobacterium-mediated expression in Nicotiana benthamiana, in pGEM-4Z (as full-length or as fusion with transit peptide of AtGWD1) for in vitro transcription, translation and chloroplast import assays
lacking nucleotides 1-225 (encoding amino acids 1-75: putative chloroplast transit peptide) in pGEM-T Easy or in pET23b for expression with C-terminal hexa-His-tag in Escherichia coli BL21(DE3)
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the starch-binding domain, SBD, Asp68-Gly184, of glucan, water dikinase 3 is cloned into the vector pET-28a+ for expression in Escherichia coli BL21DE3 cells, the region encoding residues 1-201, containing the chloroplast transit peptide and the SBD is fused to YFP and transiently expressed in Nicotiana benthamiana
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
the SEX1 gene expression is induced in HsPIPKIalpha lines, i.e. transgenic Arabidopsis thaliana plants constitutively expressing human phosphatidylinositol 4-phosphate 5-kinase Ialpha, HsPIPKIalpha, in plasma membranes. Transcripts of genes involved in starch metabolism such as SEX1 (glucan water dikinase) and SEX4 (phosphoglucan phosphatase), DBE (debranching enzyme), MEX1 (maltose transporter), APL3 (ADP-glucose pyrophosphorylase) and glucose-6-phosphate transporter (Glc6PT) are upregulated in the HsPIPKIalpha plants, overview
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
biotechnology
the starch-phosphorylating enzymes are attractive candidates for the control of flux through starch degradation
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Ritte, G.; Steup, M.; Kossmann, J.; Lloyd James, R.
Determination of the starch-phosphorylating enzyme activity in plant extracts
Planta
216
798-801
2003
Arabidopsis thaliana, Solanum tuberosum
Kotting, O.; Pusch, K.; Tiessen, A.; Geigenberger, P.; Steup, M.; Ritte, G.
Identification of a novel enzyme required for starch metabolism in Arabidopsis leaves. The phosphoglucan, water dikinase
Plant Physiol.
137
242-252
2005
Arabidopsis thaliana
Sherameti, I.; Shahollari, B.; Venus, Y.; Altschmied, L.; Varma, A.; Oelmueller, R.
The endophytic fungus Piriformospora indica stimulates the expression of nitrate reductase and the starch-degrading enzyme glucan-water dikinase in tobacco and Arabidopsis roots through a homeodomain transcription factor that binds to a conserved motif in their promoters
J. Biol. Chem.
280
26241-26247
2005
Arabidopsis thaliana, Nicotiana tabacum
Baunsgaard, L.; Lutken, H.; Mikkelsen, R.; Glaring, M.A.; Pham, T.T.; Blennow, A.
A novel isoform of glucan, water dikinase phosphorylates pre-phosphorylated alpha-glucans and is involved in starch degradation in Arabidopsis
Plant J.
41
595-605
2005
Arabidopsis thaliana
Yano, R.; Nakamura, M.; Yoneyama, T.; Nishida, I.
Starch-related alpha-glucan/water dikinase is involved in the cold-induced development of freezing tolerance in Arabidopsis
Plant Physiol.
138
837-846
2005
Arabidopsis thaliana
Ritte, G.; Heydenreich, M.; Mahlow, S.; Haebel, S.; Koetting, O.; Steup, M.
Phosphorylation of C6- and C3-positions of glucosyl residues in starch is catalysed by distinct dikinases
FEBS Lett.
580
4872-4876
2006
Arabidopsis thaliana
Edner, C.; Li, J.; Albrecht, T.; Mahlow, S.; Hejazi, M.; Hussain, H.; Kaplan, F.; Guy, C.; Smith, S.M.; Steup, M.; Ritte, G.
Glucan, water dikinase activity stimulates breakdown of starch granules by plastidial beta-amylases
Plant Physiol.
145
17-28
2007
Arabidopsis thaliana, Solanum tuberosum
Glaring, M.A.; Zygadlo, A.; Thorneycroft, D.; Schulz, A.; Smith, S.M.; Blennow, A.; Baunsgaard, L.
An extra-plastidial alpha-glucan, water dikinase from Arabidopsis phosphorylates amylopectin in vitro and is not necessary for transient starch degradation
J. Exp. Bot.
58
3949-3960
2007
Arabidopsis thaliana (Q9STV0), Arabidopsis thaliana
Hejazi, M.; Fettke, J.; Haebel, S.; Edner, C.; Paris, O.; Frohberg, C.; Steup, M.; Ritte, G.
Glucan, water dikinase phosphorylates crystalline maltodextrins and thereby initiates solubilization
Plant J.
55
323-334
2008
Arabidopsis thaliana
Christiansen, C.; Abou Hachem, M.; Janecek, S.; Viks?-Nielsen, A.; Blennow, A.; Svensson, B.
The carbohydrate-binding module family 20--diversity, structure, and function
FEBS J.
276
5006-5029
2009
Arabidopsis thaliana
Christiansen, C.; Hachem, M.A.; Glaring, M.A.; Viks?-Nielsen, A.; Sigurskjold, B.W.; Svensson, B.; Blennow, A.
A CBM20 low-affinity starch-binding domain from glucan, water dikinase
FEBS Lett.
583
1159-1163
2009
Arabidopsis thaliana
Hejazi, M.; Steup, M.; Fettke, J.
The plastidial glucan, water dikinase (GWD) catalyses multiple phosphotransfer reactions
FEBS J.
279
1953-1966
2012
Arabidopsis thaliana, Solanum tuberosum
Mahlow, S.; Orzechowski, S.; Fettke, J.
Starch phosphorylation: insights and perspectives
Cell. Mol. Life Sci.
73
2753-2764
2016
Solanum tuberosum (Q9AWA5), Arabidopsis thaliana (Q9SAC6), Arabidopsis thaliana (Q9STV0)
Mahlow, S.; Hejazi, M.; Kuhnert, F.; Garz, A.; Brust, H.; Baumann, O.; Fettke, J.
Phosphorylation of transitory starch by alpha-glucan, water dikinase during starch turnover affects the surface properties and morphology of starch granules
New Phytol.
203
495-507
2014
Arabidopsis thaliana (Q9SAC6), Arabidopsis thaliana
Skeffington, A.W.; Graf, A.; Duxbury, Z.; Gruissem, W.; Smith, A.M.
Glucan, water dikinase exerts little control over starch degradation in Arabidopsis leaves at night
Plant Physiol.
165
866-879
2014
Arabidopsis thaliana (Q9SAC6)
Hejazi, M.; Mahlow, S.; Fettke, J.
The glucan phosphorylation mediated by alpha-glucan, water dikinase (GWD) is also essential in the light phase for a functional transitory starch turn-over
Plant Signal. Behav.
9
e28892
2014
Arabidopsis thaliana (Q9SAC6)
Im, Y.J.; Smith, C.M.; Phillippy, B.Q.; Strand, D.; Kramer, D.M.; Grunden, A.M.; Boss, W.F.
Increasing phosphatidylinositol (4,5)-bisphosphate biosynthesis affects basal signaling and chloroplast metabolism in Arabidopsis thaliana
Plants (Basel)
3
27-57
2014
Arabidopsis thaliana (Q9SAC6)
Pirone, C.; Gurrieri, L.; Gaiba, I.; Adamiano, A.; Valle, F.; Trost, P.; Sparla, F.
The analysis of the different functions of starch-phosphorylating enzymes during the development of Arabidopsis thaliana plants discloses an unexpected role for the cytosolic isoform GWD2
Physiol. Plant.
160
447-457
2017
Arabidopsis thaliana (Q9SAC6), Arabidopsis thaliana (Q9STV0), Arabidopsis thaliana
Adegbaju, M.S.; Morenikeji, O.B.; Borrego, E.J.; Hudson, A.O.; Thomas, B.N.
Differential evolution of alpha-glucan water dikinase (GWD) in plants
Plants (Basel)
9
1101
2020
Amaranthus hypochondriacus, Brassica rapa, Carica papaya, Coffea arabica, Fragaria vesca, Gossypium hirsutum, Hordeum vulgare, Linum usitatissimum, Malus domestica, Physcomitrium patens, Porphyra umbilicalis, Sphagnum magellanicum, Triticum aestivum, Vigna unguiculata, Citrus clementina, Selaginella moellendorffii, Capsella rubella, Brachypodium distachyon, Chromochloris zofingiensis, Dioscorea alata, Amborella trichopoda, Malcolmia maritima, Myagrum perfoliatum, Musa acuminata subsp. malaccensis, Theobroma cacao (A0A061FDU7), Auxenochlorella protothecoides (A0A087SJ57), Solanum chacoense (A0A0V0IZQ3), Ananas comosus (A0A199UE45), Zea mays (A0A1D6LTL9), Cucumis melo (A0A1S3BEF3), Nicotiana tabacum (A0A1S3YFK2), Helianthus annuus (A0A251T3N7), Capsicum annuum (A0A2G2YEX8), Chlamydomonas reinhardtii (A0A2K3DIY0), Marchantia polymorpha (A0A2R6X3K3), Panicum miliaceum (A0A3L6S324), Panicum miliaceum, Solanum lycopersicum (B5B3R3), Sorghum bicolor (C5Z316), Vitis vinifera (D7TDL2), Glycine max (I1KXC2), Solanum tuberosum (Q9AWA5), Arabidopsis thaliana (Q9STV0), Chondrus crispus (R7QKK2), Phaseolus vulgaris (V7C6L3), Manihot esculenta (V9K6M5), Oryza sativa Japonica Group (XM_015787980.2), Ricinus communis (XP_015579774.1)
Xu, X.; Dees, D.; Huang, X.; Visser, R.; Trindade, L.
Heterologous expression of two Arabidopsis starch dikinases in potato
Starch
70
1600324
2018
Arabidopsis thaliana (Q6ZY51), Arabidopsis thaliana (Q9STV0)
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EXTERNAL LINKS (specific for EC-Number 2.7.9.4)
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