Information on EC 3.6.4.B10 - chaperonin ATPase (protein-folding, protecting from aggregation, protein stabilizing)

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

EC NUMBER
COMMENTARY hide
3.6.4.B10
preliminary BRENDA-supplied EC number
RECOMMENDED NAME
GeneOntology No.
chaperonin ATPase (protein-folding, protecting from aggregation, protein stabilizing)
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + H2O = ADP + phosphate
show the reaction diagram
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Q9YDK6: alpha-subunit, Q9YA66: beta-subunit
Q9YDK6 and Q9YA66
SwissProt
Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
gene CPKB
SwissProt
Manually annotated by BRENDA team
alpha-subunit
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + H2O
ADP + phosphate
show the reaction diagram
CTP + H2O
CDP + phosphate
show the reaction diagram
GTP + H2O
GDP + phosphate
show the reaction diagram
UTP + H2O
UDP + 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 + H2O
ADP + phosphate
show the reaction diagram
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
-
activates
Na+
activates; activates
NH4+
activates; activates
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
prefoldin
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inhibits the GFP protein refolding by interacting with intermediates
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
prefoldin
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
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kinetic analysis, stopped-flow fluorometry and stopped-flow small-angle X-ray scattering and ATP dissociation constants of wild-type and mutant enzymes. Allosteric communication in the group II chaperonins lies only in the intra-ring contact regions, which are located entirely within the equatorial domains. No cooperativity in ATP binding; kinetic analysis, stopped-flow fluorometry and stopped-flow small-angle X-ray scattering and ATP dissociation constants of wild-type and mutant enzymes. Allosteric communication in the group II chaperonins lies only in the intra-ring contact regions, which are located entirely within the equatorial domains. No cooperativity in ATP binding
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5
ATPase assay at; ATPase assay at
7.1
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assay at
7.5 - 8
thermal protection assay at; thermal protection assay at
8
chaperonin assay at; chaperonin assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50
mutants Q533G and E530G
60 - 70
wild-type enzyme and mutant P538G
70
mutants P533M, D545M, and E530M
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 90
activity range of wild-type and mutant enzymes, profiles overview
70 - 85
70C: about 60% of maximal actiovity, 85C: about 40% of maximal activity
70 - 95
70C: about 70% of maximal activity, 95C: about 50% of maximal activity
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
-
Manually annotated by BRENDA team
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
55000
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10 * 55000, SDS-PAGE, assembles into a large ring-shaped oligomeric complex comprising about 10 subunits
59140
x * 59140, calculatzed from sequence
60700
x * 60700 (subunit alpha) + x * 61200 (subunit beta), SDS-PAGE; x * 60700 + x * 61200, alpha- and beta-subunits ApCpnA and ApCpnB, respectively, SDS-PAGE; x * 60700 + x * 61200, alpha- and beta-subunits ApCpnA and ApCpnB, respectively, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
decamer
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10 * 55000, SDS-PAGE, assembles into a large ring-shaped oligomeric complex comprising about 10 subunits
hexadecamer
octamer
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unique asymmetric pattern among the eight subunits of TRiC in the nucleotide-free state, mechanism of TRiC negative inter-ring cooperativity. The TRiC lid remains open in three states of the cycle: ATP bound, ADP bound, and nucleotide free, structure-function relationship, structure modeling and electron cryo-microscopy, overview
oligomer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure analysis, PDB ID 3KFK
crystal structure analysis, PDB IDs 3IYF and 3LOS, enzyme in open and closed state conformation
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crystal structure analysis, PDB ID 4D8Q
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
each of the recombinant alpha- and beta- subunits is purified to 92% and 94% by using anion exchange chromatography
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recombinant alpha-subunit from Escherichia coli strains Rosetta (DE3), BL21 (DE3), or CodonPlus (DE3) by heat shock treatment and anion exchange chromatography; recombinant beta-subunit from Escherichia coli strains Rosetta (DE3), BL21 (DE3), or CodonPlus (DE3) by heat shock treatment and anion exchange chromatography; recombinant subunit alpha and subunit beta
recombinant enzyme
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recombinant enzyme CPN from Escherichia coli strain BLR(DE3)/pET23a by heat treatment for 15 min at 90C, anion exchange chromatography, ultrafiltration, and gel filtration
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recombinant His-tagged wild-type and mutant enzyme oligomer rings from Escherichia coli; recombinant His-tagged wild-type and mutant enzyme oligomer rings from Escherichia coli
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) RIL by heat treatment at 85C for 30 min, anion exchange chromatography, and gel filtration
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ApCpnA and ApCpnB chaperonin genes are overexpressed in Escherichia coli Rosetta and Codonplus (DE3), respectively
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expressed in Escherichia coli
expression in Escherichia coli
expression of ApCpnA (subunit alpha) and ApCpnB (subunit beta) in Escherichia coli Rosetta (DE3), BL21 (DE3), or CodonPlus (DE3) cells; gene thsA, recombinant expression of alpha-subunit in Escherichia coli strains Rosetta (DE3), BL21 (DE3), or CodonPlus (DE3); gene thsB, recombinant expression of beta-subunit in Escherichia coli strains Rosetta (DE3), BL21 (DE3), or CodonPlus (DE3)
gene cpkA, recombinant expression of the chimeric chaperonin CpkBA in Escherichia coli; gene cpkB, recombinant expression of the chimeric chaperonin CpkBA in Escherichia coli
recombinant expression of enzyme CPN in Escherichia coli strain BLR(DE3)/pET23a
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recombinant expression of His-tagged wild-type and mutant enzyme oligomer rings in Escherichia coli; recombinant expression of His-tagged wild-type and mutant enzyme oligomer rings in Escherichia coli
recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) RIL
subcloned into vector pET21a. The constructed pET21a-ApCpnB is transformed into Escherichia coli BL21 Codonplus (DE3)
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
induced when halophilic cells are exposed to a low salt stress
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possesses two chaperonins, cold-inducible CpkA and heat-inducible CpkB, which are involved in adaptation to low and high temperatures, respectively
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possesses two chaperonins, cold-inducible CpkA and heat-inducible CpkB, which are involved in adaptation to low and high temperatures, respectively; possesses two chaperonins, cold-inducible CpkA and heat-inducible CpkB, which are involved in adaptation to low and high temperatures, respectively
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
G160S
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the TRiC-like mutant G160S of MmCpn has a drastically slower rate of ATP hydrolysis, roughly equivalent to the steady-state hydrolysis of eukaryotic TRiC
D545G
site-directed mutagenesis, the mutant shows a stabilities similar to the wild-type CpkA
D545M
site-directed mutagenesis, the mutant shows slightly higher stabilities than that of wild-type CpkA
E530G
site-directed mutagenesis, the mutant strain DA4 shows increased ATPase activity. The CpkA-E530G mutation prevents cold denaturation of proteins under cold-stress conditions, thereby enabling cells to grow in cooler environments
E530M
site-directed mutagenesis, the mutant shows a stabilities similar to the wild-type CpkA
P538G
site-directed mutagenesis, the mutant shows slightly higher stabilities than that of wild-type CpkA
P538M
site-directed mutagenesis, the mutant shows a stabilities similar to the wild-type CpkA
Q533G
site-directed mutagenesis
Q533M
site-directed mutagenesis, the mutant shows a stabilities similar to the wild-type CpkA
D64A/D393A
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site-directed mutagenesis, an ATPase-deficient mutant, the mutant also does not exhibit ATPase-dependent conformational change; site-directed mutagenesis, an ATPase-deficient mutant, the mutant also does not exhibit ATPase-dependent conformational change
D64A/D393A/K485W
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site-directed mutagenesis, an ATPase-deficient mutant, the mutant also does not exhibit ATPase-dependent conformational change, the mutant lacks ATP-dependent refolding activity, nucleotide binding and ATP-dependent conformational change kinetics, overview; site-directed mutagenesis, an ATPase-deficient mutant, the mutant also does not exhibit ATPase-dependent conformational change, the mutant lacks ATP-dependent refolding activity, nucleotide binding and ATP-dependent conformational change kinetics, overview
K165A/K485W
site-directed mutagenesis, ATPase inactive mutant that can partially prevent the spontaneous refolding ofGFP and refold it in an ATP-dependent manner; site-directed mutagenesis, ATPase inactive mutant that can partially prevent the spontaneous refolding ofGFP and refold it in an ATP-dependent manner
L265W
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site-directed mutagenesis, replacement of amino acid L265 with Trp partially impairs the protein folding activity, eight Trp residues are thought to come close in the closed conformation. The resulting steric hindrance might interrupt the conformational changes required for protein folding. Although ATP hydrolysis activity is almost completely lost in the absence of K+, slight ATP-dependent folding activity is observed; site-directed mutagenesis, replacement of amino acid L265 with Trp partially impairs the protein folding activity, eight Trp residues are thought to come close in the closed conformation. The resulting steric hindrance might interrupt the conformational changes required for protein folding. Although ATP hydrolysis activity is almost completely lost in the absence of K+, slight ATP-dependent folding activity is observed
L56W
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site-directed mutagenesis, the mutant exhibits nearly the same level of protein folding activity as the wild-type protein; site-directed mutagenesis, the mutant exhibits nearly the same level of protein folding activity as the wild-type protein
K165A/K485W
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site-directed mutagenesis, ATPase inactive mutant that can partially prevent the spontaneous refolding ofGFP and refold it in an ATP-dependent manner
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K485W
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site-directed mutagenesis, the mutant shows ATP binding and conformational change upon ATP binding
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additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
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ApCpnB has both foldase and holdase activities and can be used as a powerful molecular machinery for the production of recombinant proteins as soluble and active forms in Escherichia coli