Information on EC 3.4.15.6 - cyanophycinase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.4.15.6
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RECOMMENDED NAME
GeneOntology No.
cyanophycinase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
[L-Asp(4-L-Arg)]n + H2O = [L-Asp(4-L-Arg)]n-1 + L-Asp(4-L-Arg)
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
cyanophycin metabolism
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CAS REGISTRY NUMBER
COMMENTARY hide
131554-16-0
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain ADP1
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Manually annotated by BRENDA team
strain ADP1
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-
Manually annotated by BRENDA team
strain ATCC 29413
UniProt
Manually annotated by BRENDA team
strain CB15
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-
Manually annotated by BRENDA team
strain CB15
-
-
Manually annotated by BRENDA team
strain J-10-fl
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-
Manually annotated by BRENDA team
strain J-10-fl
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-
Manually annotated by BRENDA team
strain DSM3043
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-
Manually annotated by BRENDA team
strain DSM3043
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-
Manually annotated by BRENDA team
strain NCIMB 8052
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-
Manually annotated by BRENDA team
strain NCIMB 8052
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-
Manually annotated by BRENDA team
strain a
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-
Manually annotated by BRENDA team
strains 13, SM101, and ATCC13124
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-
Manually annotated by BRENDA team
strain E88
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-
Manually annotated by BRENDA team
strain E88
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-
Manually annotated by BRENDA team
strain 34H
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-
Manually annotated by BRENDA team
strain WH 8501
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-
Manually annotated by BRENDA team
strain WH 8501
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-
Manually annotated by BRENDA team
strain H16
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-
Manually annotated by BRENDA team
strain H16
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-
Manually annotated by BRENDA team
strain DCB-2
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-
Manually annotated by BRENDA team
strain DCB-2
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-
Manually annotated by BRENDA team
Francisella tularensis tularensis, strain Schu 4
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-
Manually annotated by BRENDA team
strain UQM 2246
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-
Manually annotated by BRENDA team
strain UQM 2246
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Manually annotated by BRENDA team
strain PCC 7421
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Manually annotated by BRENDA team
strain HTCC2649
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Manually annotated by BRENDA team
strain HTCC2649
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Manually annotated by BRENDA team
strain M
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-
Manually annotated by BRENDA team
strain PCC 73102
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-
Manually annotated by BRENDA team
strain JS666
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-
Manually annotated by BRENDA team
strain JS666
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-
Manually annotated by BRENDA team
strain BI
SwissProt
Manually annotated by BRENDA team
strain PM1
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Manually annotated by BRENDA team
strain PM1
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Manually annotated by BRENDA team
strain ATCC 27405
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Manually annotated by BRENDA team
strains PCC 7002 and PCC 6803
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-
Manually annotated by BRENDA team
strain PCC 6803
SwissProt
Manually annotated by BRENDA team
strain Goettingen
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-
Manually annotated by BRENDA team
strain Goettingen
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Manually annotated by BRENDA team
strain ATCC 33223
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Manually annotated by BRENDA team
strain BP-1
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Manually annotated by BRENDA team
strain IMS101
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
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compartmentalized degradation of cyanophycin, with the first step, catalyzed by cyanophycinase, taking place in the heterocysts and the second step, catalyzed by a dipeptidase, taking place mainly in the vegetative cells
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
modified soluble cyanobacterial granule polypeptide + H2O
beta-aspartate-arginine + beta-aspartate-homoarginine + ?
show the reaction diagram
[L-Asp(4-Arg)]n + H2O
[L-Asp(4-Arg)]n-1 + [L-Asp(4-Arg)]
show the reaction diagram
[L-Asp(4-L-Arg)]n + H2O
[L-Asp(4-L-Arg)]n-1 + [L-Asp(4-L-Arg)]
show the reaction diagram
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
[L-Asp(4-Arg)]n + H2O
[L-Asp(4-Arg)]n-1 + [L-Asp(4-Arg)]
show the reaction diagram
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3,4-dichloro-isocoumarin
78% inhibition at 1 mM, 56% inhibition at 0.1 mM
4-(2-aminoethyl) benzenesulfonylfluoride
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cyanophycinase is strongly inhibited
EDTA
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49% inhibition at 60 nM, 46% inhibition at 30 nM
leupeptin
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13% inhibition within the range 0.001 nM to 0.01 nM
N-bromo-succinimide
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100% inhibition at 5 nM, 99% inhibition at 1 nM
N-bromosuccinimide
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total inhibition
pefabloc
phenylmethanesulfonyl fluoride
45% inhibition at 10 mM, 8% inhibition at 1 mM
phenylmethylsulfonyl fluoride
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0022
[L-Asp(4-Arg)]n
30°C in 50 mM N-[2-hydroxy-1,1-bis(hydroxymethyl) ethyl]glycine, pH 8.0
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
16.5
[L-Asp(4-Arg)]n
Synechocystis sp. PCC 6803
P73832
30°C in 50 mM N-[2-hydroxy-1,1-bis(hydroxymethyl) ethyl]glycine, pH 8.0, maximum at pH 9.0
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
7500
[L-Asp(4-Arg)]n
Synechocystis sp. PCC 6803
P73832
30°C in 50 mM N-[2-hydroxy-1,1-bis(hydroxymethyl) ethyl]glycine, maximum at pH 8.0
35296
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
broad pH maximum between pH 7 and pH 8, peaking at pH 7.5
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 9
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20 min, 30°C
6
30% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10 - 80
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20 min, sodium phosphate buffer pH 7.0
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.92
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calculated from the sequence
6.51
calculated from sequence
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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compartmentalized degradation of cyanophycin, with the first step, catalyzed by cyanophycinase, taking place in the heterocysts and the second step, catalyzed by a dipeptidase, taking place mainly in the vegetative cells
Manually annotated by BRENDA team
additional information
cyanophycinase is amplified from Synechocystis sp. PCC 6803 genomic DNA using iProof polymerase and is ligated into the BamHI and NdeI sites of pET-28a, plasmids are transformed into Rosetta cells
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Synechocystis sp. (strain PCC 6803 / Kazusa)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
27000
1 * 27000, SDS-PAGE
28000
gel filtration
29700
2 * 29700, SDS-PAGE
43000
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monomer, SDS-PAGE
45000
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SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
2 * 29700, SDS-PAGE
monomer
1 * 27000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
12 mg/ml protein, 1.6 M ammonium sulfate, 0.1 M MES, 12% v/v dioxane, pH 6.6, vapor diffusion, sitting drop, temperature 293 K, space group C2221, resolution 1.5 A, the native crystal is grown in the presence of 5 mM cyanophycinase product, but no electron density for the product is interpretable in the final map
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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maximum degradation activity at 50°C and complete inactivation at 68°C
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
centrifugation, proteins are concentrated and desalted using a cross-flow polyethersulfone cassette with a cutoff point of 30 kDa, a purification method for cyanophycinase is developed which depended on the strong affinity of the enzyme in the crude extract for its insoluble substrate cyanophycin
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from culture supernatant of Pseudomonas anguilliseptica cells grown in medium containing cyanophycin granules, anion-exchange chromatography and L-arginine-agarose affinity chromatography. In case of heterologous expression in Escherichia coli the enzyme was not secreted and thus, isolation was done from the cell lysate
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
expression in Escherichia coli DH5alpha
expression in Escherichia coli XL1-Blue
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
N100A
site-directed mutagenesis, 40% activity of the wild-type, measured with 75 microg/ml of [L-Asp(4-Arg)]n in phosphate buffer at pH 8.3 and 30°C
N158A
site-directed mutagenesis, 41% activity of the wild-type, measured with 75 microg/ml of [L-Asp(4-Arg)]n in phosphate buffer at pH 8.3 and 30°C
N172A
site-directed mutagenesis, 0.02% activity of the wild-type, measured with 75 microg/ml of [L-Asp(4-Arg)]n in phosphate buffer at pH 8.3 and 30°C
N17A
site-directed mutagenesis, 60% activity of the wild-type, measured with 75 microg/ml of [L-Asp(4-Arg)]n in phosphate buffer at pH 8.3 and 30°C
N202A
site-directed mutagenesis, 8% activity of the wild-type, measured with 75 microg/ml of [L-Asp(4-Arg)]n in phosphate buffer at pH 8.3 and 30°C
Q101A
site-directed mutagenesis, 0.02% activity of the wild-type, measured with 75 microg/ml of [L-Asp(4-Arg)]n in phosphate buffer at pH 8.3 and 30°C
Q173A
site-directed mutagenesis, 0.5% activity of the wild-type, measured with 75 microg/ml of [L-Asp(4-Arg)]n in phosphate buffer at pH 8.3 and 30°C
R178A
site-directed mutagenesis, 1% activity of the wild-type, measured with 75 microg/ml of [L-Asp(4-Arg)]n in phosphate buffer at pH 8.3 and 30°C
R180A
site-directed mutagenesis, 0.01% activity of the wild-type, measured with 75 microg/ml of [L-Asp(4-Arg)]n in phosphate buffer at pH 8.3 and 30°C
R183a
site-directed mutagenesis, 0.01% activity of the wild-type, measured with 75 microg/ml of [L-Asp(4-Arg)]n in phosphate buffer at pH 8.3 and 30°C
S132A
site-directed mutagenesis, 0.002% activity of the wild-type, measured with 75 microg/ml of [L-Asp(4-Arg)]n in phosphate buffer at pH 8.3 and 30°C
additional information
Gly101, Asp172, Gly173, Arg178, Arg180 and Arg183 form a conserved pocket adjacent to the catalytic Ser132, these are functionally critical residues. The cyanophycinase active site contains three absolutely conserved Arg residues (Arg178, Arg180 and Arg183), which line up along one side of the pocket
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
in-gel renaturation method
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
food industry
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