Information on EC 2.7.9.4 - alpha-glucan, water dikinase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.7.9.4
-
RECOMMENDED NAME
GeneOntology No.
alpha-glucan, water dikinase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + alpha-glucan + H2O = AMP + phospho-alpha-glucan + phosphate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
esterification
-
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methyl group transfer
-
-
-
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Phosphorylation
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-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
starch degradation II
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-
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].
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
cv. Golden Promise
UniProt
Manually annotated by BRENDA team
AtGWD1, AtGWD2 and AtGWD3 ORFs, nucleotide-binding domain; genotype TMS 4(2)1425
D7RA57 AND D7RA56
UniProt
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
Araceae
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
metabolism
physiological function
additional information
potato lines GWD phenotype-genotype relationship, ooverview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
amylopectin + ATP + H2O
AMP + phospho-amylopectin + phosphate
show the reaction diagram
ATP + 6-O-alpha-maltosyl-beta-cyclodextrin + H2O
AMP + phospho-6-O-alpha-maltosyl-beta-cyclodextrin + phosphate
show the reaction diagram
-
-
-
-
?
ATP + alpha-cyclodextrin + H2O
AMP + phospho-alpha-cyclodextrin + phosphate
show the reaction diagram
-
-
-
-
?
ATP + alpha-glucan + H2O
AMP + phospho-alpha-glucan + phosphate
show the reaction diagram
ATP + amylopectin + H2O
?
show the reaction diagram
-
phosphorylation at the C3 position of glucose residues in amylopectin
-
-
?
ATP + amylopectin + H2O
AMP + phospho-amylopectin + phosphate
show the reaction diagram
ATP + amylose 18 + H2O
AMP + phosphorylated amylose 18 + phosphate
show the reaction diagram
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-
very poor substrate
-
?
ATP + amylose 24 + H2O
AMP + phosphorylated amylose 24 + phosphate
show the reaction diagram
-
-
very poor substrate
-
?
ATP + amylose 53 + H2O
AMP + phosphorylated amylose 53 + phosphate
show the reaction diagram
-
-
-
-
?
ATP + amylose 85 + H2O
AMP + phosphorylated amylose 85 + phosphate
show the reaction diagram
-
-
-
-
?
ATP + beta-cyclodextrin + H2O
AMP + phospho-beta-cyclodextrin + phosphate
show the reaction diagram
-
-
-
-
?
ATP + crystalline maltodextrin + H2O
AMP + phospho-alpha-glucosyl-maltodextrin + phosphate
show the reaction diagram
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
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-
?
ATP + crystalline maltodextrin + H2O
AMP + phosphorylated crystalline maltodextrin + phosphate
show the reaction diagram
ATP + elongated glucogen + H2O
AMP + elongated phosphoglucogen + phosphate
show the reaction diagram
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-
-
-
?
ATP + granular potato starch + H2O
AMP + phosphorylated granular potato starch + phosphate
show the reaction diagram
-
-
-
-
?
ATP + maltodextrin + H2O
?
show the reaction diagram
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crystallized maltodextrins, A- and B-type allomorphs
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-
?
ATP + postelongated glycogen + H2O
AMP + postelongated phospho-glycogen + phosphate
show the reaction diagram
-
-
-
-
?
ATP + potato amylopektin + H2O
AMP + phosphorylated potato amylopektin + phosphate
show the reaction diagram
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-
-
-
?
ATP + potato amylose + H2O
AMP + phosphorylated potato amylose + phosphate
show the reaction diagram
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-
-
-
?
ATP + starch + H2O
AMP + phosphorylated starch + phosphate
show the reaction diagram
phospho-amylopectin + ATP + H2O
AMP + phospho-amylopectin + phosphate
show the reaction diagram
starch + ATP + H2O
AMP + phospho-starch + phosphate
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
amylopectin + ATP + H2O
AMP + phospho-amylopectin + phosphate
show the reaction diagram
ATP + alpha-glucan + H2O
AMP + phospho-alpha-glucan + phosphate
show the reaction diagram
ATP + amylopectin + H2O
?
show the reaction diagram
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phosphorylation at the C3 position of glucose residues in amylopectin
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-
?
phospho-amylopectin + ATP + H2O
AMP + phospho-amylopectin + phosphate
show the reaction diagram
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the enzyme is involved in degradation of transistory starch
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-
?
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
soluble maltodextrin
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-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
thioredoxin
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starch granule-bound enzyme, isolated from dark-adapted plants exists in the inactive, oxidized form, which is capable of reactivation upon treatment with reduced thioredoxin
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00023
ATP
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.015
granular potato starch
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30C
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00007
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wheat starch, kernel, A-allomorph
0.00008
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waxy maize starch, kernel, A-allomorph
0.00011
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maize starch, kernel, A-allomorph
0.0012
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amylose extender mutant maize, kernel, B-allomorph
0.0023
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potato starch, tuber, B-allomorph
0.0112
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Arabidopsis sex1-3, leaf, B-allomorph
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
0.058
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crystalline maltodextrin, B-allomorph
0.07
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30C, wild-type, substrate potato amylose
0.14
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30C, fragment N11, substrate potato amylose
0.2
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30C, wild-type, substrate amylose 53
0.41
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30C, wild-type, substrate amylose 85
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
1.01
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30C, fragment N11, substrate amylose 53
1.18
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30C, fragment N11, substrate amylose 85
1.36
soluble potato starch
3.26
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30C, wild-type, substrate potato amylopektin
5.8
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30C, wild-type, substrate granular potato starch
6.19
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30C, fragment N11, substrate potato amylopektin
additional information
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conversion of heat solubilized maltodextrin below 1 nmol/min/mg
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.4
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assay at
7.5
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activity assay
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 40
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20C: about 75% of maximal activity, 40C: about 85% of maximal activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
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phosphorylated GWD
5.5
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non-phosphorylated GWD
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
floral organ, strong expression in vascular tissue just before onset of senescence (promoter activity)
Manually annotated by BRENDA team
high expression level (microarray)
Manually annotated by BRENDA team
phloem, high expression just before onset of senescence (promoter activity)
Manually annotated by BRENDA team
strong expression in mature flower parts (microarray)
Manually annotated by BRENDA team
in vitro germination at 25C for 8 h (to 50% impaired in absence of functional GWD, starch accumulation and reduction in level of soluble sugars)
Manually annotated by BRENDA team
strong expression in vascular tissue just before onset of senescence (promoter activity)
Manually annotated by BRENDA team
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70% of the total R1 protein found in the soluble fraction, strong increase of soluble enzyme activity during continuous red light
Manually annotated by BRENDA team
strong expression in vascular tissue just before onset of senescence (promoter activity)
Manually annotated by BRENDA team
leaf, stem, root, flower, silique only just before onset of senescence (promoter activity)
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
along the cell wall, N-terminal 158 and 61 amino acids (revealed by confocal laser scanning microscopy)
Manually annotated by BRENDA team
the expression of AtGWD2 is in a relatively late stage of plant development
Manually annotated by BRENDA team
N-terminal 158 and 61 amino acids (revealed by confocal laser scanning microscopy)
Manually annotated by BRENDA team
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starch granule-bound enzyme, isolated from dark-adapted plants exists in the inactive, oxidized form, which is capable of reactivation upon treatment with reduced thioredoxin
Manually annotated by BRENDA team
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
14200
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theoretical, starch-binding domain, SBD, Asp68-Gly184, verified by SDS-PAGE
152000
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multi-angle laser light scattering after gel-filtration
154000
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starch-associated, phosphorylated and non-phosphorylated GWD, SDS-PAGE
155000
x * 155000, SDS-PAGE
160000
330000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
D7RA57 AND D7RA56
the GWD protein consists of a dikinase domain and a large (approximately 120 kDa) N-terminal domain
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phosphoprotein
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
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5 min, about 15% loss of activity
50
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5 min, about 40% loss of activity
55
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5 min, complete loss of activity
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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extracted from starch granules with SDS, washed with methanol and chloroform and identified by 2D-SDS-PAGE and immunodetection
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HisTrap column chromatography and Mono Q column chromatography
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Ni-nitrilotriacetic acid column chromatography, and gel filtration
recombinant enzyme, homogenous
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recombinant enzyme, partially
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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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wild-type and mutant enzyme H992A
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
amplified from young leave cDNA for sequencing; from pBluescript-GWD via pAA100-35S and pBinPlus to construct pLAT52::StGWD for expression in Micro-Tom tomato plants under control of a late pollen-specific promoter following Agrobacterium-mediated transformation
expressed in Escherichia coli
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expressed in Escherichia coli BL21 cells and in Nicotiana tabacum leaf mesophyll cells
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expressed in Escherichia coli BL21(DE3) cells
expression in Escherichia coli
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expression in Saccharomyces cerevisiae YWO 0046
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gene GWD, genotyping included determination of allele dosage of 193 potato lines by amplicon sequencing and microsatellite analysis, overview
gene gwd, recombinant expression of Arabidopsis thaliana GWD mutant C1019S C-terminally fused to GFP from a 35S promoter in transgenic Arabidopsis thaliana plants
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gene GWD1, construction of transgenic plants expressing the soluble StrepII-tagged mutant enzyme, transformation via Agrobacterium tumefaciens mediation method
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gene GWD1, the gene is located on chromosome 1, phylogenetic tree; gene GWD2
gene R1 or GWD, DNA and amino acid sequence determination and analysis by reverse transcription PCR approach using tuber RNA, detailed sequence comparison and phylogenetic analysis, semi-quantitative RT-PCR gene expression analysis
genotype TMS 4(2)1425, DNA and amino acid sequence determination and analysis of the enzyme and the nucleotide-binding domain of the enzyme, sequence comparisons and phylogenetic tree
D7RA57 AND D7RA56
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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overexpression of the enzyme in Hordeum vulgare using the hordein-D promoter, the promoter activates storage protein accumulation, mostly at a later grain developmental stage than 10 DAP
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
isoform GWD1 expression reaches its maximum after 10 h of illumination, The lowest expression of isoform GWD1 occurs 4 h after turning the light on, and it is approximately 7times lower than the expression measured 6 h late
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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
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C1019S
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site-directed mutagenesis, the mutant is redox-insensitiv in contrast to the wild-type enzyme
H1004A
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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
H992A
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mutant enzyme backgroun level of activity, no autophosphorylation
W117A
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mutation in tandem repeat sequence SBD-1, mutant shows extremely weak glucan binding
W62A
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mutation in tandem repeat sequence SBD-1, mutant shows extremely weak glucan binding
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
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the starch-phosphorylating enzymes are attractive candidates for the control of flux through starch degradation