Information on EC 6.3.2.30 - cyanophycin synthase (L-arginine-adding)

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
6.3.2.30
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RECOMMENDED NAME
GeneOntology No.
cyanophycin synthase (L-arginine-adding)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + [L-Asp(4-L-Arg)]n-L-Asp + L-Arg = ADP + phosphate + [L-Asp(4-L-Arg)]n+1
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
carboxylic acid amide formation
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
cyanophycin metabolism
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-
SYSTEMATIC NAME
IUBMB Comments
cyanophycin:L-arginine ligase (ADP-forming)
Requires Mg2+ for activity. Both this enzyme and EC 6.3.2.29, cyanophycin synthase (L-aspartate-adding), are required for the elongation of cyanophycin, which is a protein-like cell inclusion that is unique to cyanobacteria and acts as a temporary nitrogen store [2]. Both enzymes are found in the same protein but have different active sites [2,4]. Both L-Asp and L-Arg must be present before either enzyme will display significant activity [2]. Canavanine and lysine can be incoporated into the polymer instead of arginine [2].
CAS REGISTRY NUMBER
COMMENTARY hide
131554-17-1
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain ADP1
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-
Manually annotated by BRENDA team
gene cphA1
UniProt
Manually annotated by BRENDA team
strain NE1
-
-
Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
cf. EC 6.3.2.29
UniProt
Manually annotated by BRENDA team
strain PCC6308
-
-
Manually annotated by BRENDA team
gene cphA49 from deep-sea sediment metagenomic library
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
cyanophycin synthetase CphA49 belongs to NOR5 clade of Gammaproteobacteria enzymes
malfunction
metabolism
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + [L-Asp(4-L-Arg)]n-L-Asp + L-Arg
ADP + phosphate + [L-Asp(4-L-Arg)]n+1
show the reaction diagram
ATP + [L-Asp(4-L-Arg)]n-L-Asp + L-Arg
[L-Asp(4-L-Arg)]n+1 + ADP + phosphate
show the reaction diagram
L-Lys cannot replace L-Arg in reaction with CphANE1 or CphANE1del96
-
-
?
L-aspartic acid + ATP
poly-L-aspartic acid + ADP + phosphate
show the reaction diagram
-
-
-
-
?
additional information
?
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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
ATP + [L-Asp(4-L-Arg)]n-L-Asp + L-Arg
ADP + phosphate + [L-Asp(4-L-Arg)]n+1
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mn2+
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Mg2+ or Mn2+ are required
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
sulfhydryl reagents
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additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.2 - 0.278
ATP
0.24 - 0.45
L-aspartic acid
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00221
at pH 8.2 and 28°C
0.113
30°C, pH 8.2, 123fold purification
0.238
after 72fold purification, 28°C, pH 8.2
0.265
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30°C, pH 8.2
2.01
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wild-type enzyme
3.2
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mutant CphA6308DELTA1
4.17
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mutant CphA6308DELTA1/C595S
4.95
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mutant C595S
17.5
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purified recombinant wild-type enzyme at 50°C
additional information
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activities of recombinant truncation mutant enzymes, overview
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6
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recombinant enzyme in Pichia pastoris strain GS115
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 11
activity range, high activity at a pH range of pH 8.0 to pH 10.5, recombinant enzyme, profile overview
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
26
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recombinant enzyme in Pichia pastoris strain GS115
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10 - 70
activity range, profile overview
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
cyanophycin is deposited in the cytoplasm as membraneless granules
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
90000
2 * 90000, SDS-PAGE
98000
x * 98000, about, sequence calculation, x * 100000, recombinant His-tagged enzyme, SDS-PAGE
98230
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x * 100000, recombinant His6-tagged wild-type enzyme, SDS-PAGE, x * 98230, His6-tagged wild-type enzyme, sequence calcualtion
100000
100600
monomer, calculated from the amino acid sequence
210000
-
gel filtration, recombinant wild-type enzyme
230000
gel filtration
240000
gel filtration
400000
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the molecular mass of cyanophycin increases with increasing reaction temperature
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tetramer
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4 * 100000, SDS-PAGE
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
28
still active after 30 min incubation
40
purified recombinant enzyme, stable up to
50 - 60
preincubation for 30 min in this temperature range results in 84% to 72% activity compared to preincubation at 30°C
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
purified enzyme is unstable at both 0°C and -70°C
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
at 7°C the purified enzyme is stable for about one week, thereafter the activity decreases rapidly, storage at -20°C in the presence of 10% (w/v) DMSO or 50% (w/v) glycerol does not improve the stability significantly
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
anion-exchange chromatography, gel filtration, affinity precipitation with cyanophycin, 69fold purification
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dye-ligand, gel filtration and ion-exchange chromatography, enriched about 4500fold
from Synechocystis and from recombinant Escherichia coli cells
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recombinant His-tagged CphA49 from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, dialysis, and ultrafiltration
recombinant His-tagged enzyme by nickel affinity chromatography and gel filtration
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recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration
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recombinant His6-tagged wild-type and mutant enzymes by His affinity chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
engineered cyanophycin synthetase (CphA) from Nostoc ellipsosporum confers enhanced CphA activity and cyanophycin accumulation to Escherichia coli
expression in Escherichia coli BL21(DE3)
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expression in Escherichia coli DH1 cells
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expression in Escherichia coli DH5alpha
expression in Escherichia coli TOP10 cells
expression in Pseudomonas putida ATCC 4359
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expression in the wild-type Sinorhizobium meliloti 1021 and in a phbC-negative mutant
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functional expression in Nicotiana tabacum var. Petit Havana SRI targeted to the chloroplasts using the CaMV 35S promoter and a translocation pathway signal sequence, the phenotypic abnormalities are reduced by this way, cyanophycin accumulation in chloroplasts, overview
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gene cphA, DNA and amino acid sequence determination and analysis, phylogenetic analysis, functional overexpression of His-tagged Tlr2170 in Escherichia coli BL21(DE3)
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gene cphA, expression in Solanum tuberosum tubers under control of the tuber-specific class 1 promoter B33 directed to tuber cytosol or to tuber plastids leading to cyanophycin accumulation in the tubers
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gene cphA, subcloning and expression of His-tagged wild-type and mutant enzymes in Escherichia coli strains DH5alpha and BL21(DE3)
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gene cphA49 cloned from deep-sea sediment metagenomic library with degenerated primers, amplified from the fss49 fosmid by PCR, DNA and amino acid sequence determination and analysis, functional expression of His-tagged cphA49 in Escherichia coli strain BL21(DE3), phylogenetic tree, subcloning in Escherichia coli strain DH5alpha
gene cphA6308, expression in Pichia pastoris and Escherichia coli from expression vectors pPICHOLI-C with the copper-inducible CUP1 promoter and pPICHOLI-3 with the methanol-inducible AOX1 promoter. Stabilization of the expression plasmid, the his4 gene from Saccharomyces cerevisiae is introduced and the expression is made in the His auxotrophic Pichia pastoris strain GS115. Recombinant polymer production by wild-type and mutant enzymes and product compositions, overview
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gene cphA6308, functional expression in Saccharomyces cerevisiae strains G175 and BY4741, which is much more efficient with the copper ion-inducible CUP1 promoter instead of the GAL1 promoter, the yeast strains produce water-soluble and water-insoluble cyanophycin polymer. Growth of transgenic yeasts in the presence of 15 mM lysine results in an incorporation of up to 10 mol% of lysine into cyanophycin, overview. Subcloning in Escherichia coli strain XL1-Blue
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gene cphA6803, enzyme expression in auxotrophic mutant Rhizopus oryzae strain M16 under control of the pyruvate decarboxylase promoter and terminator elements of Rhizopus oryzae by biolistic transformation method
gene cphA7120, enzyme expression in auxotrophic mutant Rhizopus oryzae strain M16 under control of the pyruvate decarboxylase promoter and terminator elements of Rhizopus oryzae by biolistic transformation method
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E856A
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site-directed mutagenesis, inactive mutant
E856V
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site-directed mutagenesis, inactive mutant
E856A
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site-directed mutagenesis, inactive mutant
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E856V
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site-directed mutagenesis, inactive mutant
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additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
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