Information on EC 3.6.4.5 - minus-end-directed kinesin ATPase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.6.4.5
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RECOMMENDED NAME
GeneOntology No.
minus-end-directed kinesin ATPase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + H2O = ADP + phosphate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of phosphoric ester
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-
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SYSTEMATIC NAME
IUBMB Comments
kinesin ATP phosphohydrolase (minus-end-directed)
Structurally almost identical to EC 3.6.4.3 (microtubule-severing ATPase) but the movement it catalyses is towards the minus end of microtubules.
CAS REGISTRY NUMBER
COMMENTARY hide
9000-83-3
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
squid
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Manually annotated by BRENDA team
ATCC 10895
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Manually annotated by BRENDA team
chicken, cell line HD-11
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Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
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knockdown of KifC3 using RNAi results in an increase of cells with perinuclear-clustered peroxisomes, indicating enhanced minus-end directed motility of peroxisomes leading to peroxisomal clustering at the microtubule-organizing center. The peroxisomal phenotype is cell cycle phase independent, while microtubules are essential for phenotype formation
metabolism
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
2'-dATP + H2O
2'-dADP + phosphate
show the reaction diagram
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-
-
-
?
2'-deoxy-ATP + H2O
2'-dADP + phosphate
show the reaction diagram
3'-dATP + H2O
3'-dADP + phosphate
show the reaction diagram
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-
-
-
?
3'-deoxy-ATP + H2O
3'-dADP + phosphate
show the reaction diagram
ATP + H2O
?
show the reaction diagram
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-
-
-
?
ATP + H2O
ADP + phosphate
show the reaction diagram
ddATP + H2O
2',3'-dideoxyADP + phosphate
show the reaction diagram
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-
-
-
?
dideoxy-ATP + H2O
monomethyl-ADP + phosphate
show the reaction diagram
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-
-
-
?
monomethyl-ATP + H2O
?
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
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5'-adenylylimidodiphosphate
Acrylamide
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kinesin inhibition is observed at acrylamide concentrations as low as 0.1 mM
glycidamide
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kinesin inhibition is observed at glycidamide concentrations as low as 0.1 mM
importin alpha/beta
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binding of importin alpha/beta to XCTK2 inhibits its association with microtubules and blocks its ability to cross-link microtubules
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Kar3
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Ncd and Kar3, both minus-end motors, severely inhibit the binding of one another to microtubules
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monastrol
N-ethylmaleimide
Propionamide
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weak inhibition
VO43-
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-
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Cik1
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Cik1 and Kar3 stalk regions preferentially associate with one another rather than forming homodimers. Kar3/Cik1 moves on microtubules at 0.002-0.0024 mm/min, 2-5fold faster than Kar3, and destabilizes microtubules at the lagging ends. Structural changes in Kar3 upon dimerization with Cik1 alter the motor velocity and likely regulate Kar3 activity in vivo
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microtubule
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microtubules
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stimulates ncd ATPase activity by ca. 30fold
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additional information
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microtubule-stimulated ATPase
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00014 - 0.059
ATP
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0008 - 70
ATP
additional information
ATP
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.1
calculated from amino acid sequence
10
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theoretical pI-value
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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associated with Triton X-100 insoluble membrane organelles
Manually annotated by BRENDA team
additional information
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endogenous KifC3 as well as overexpressed KifC3 cannot be detected at peroxisomes. Overall peroxisomal distribution is not affected by overexpression of either human KifC3
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Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
15000
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His6Kar3/Cik1-S tag protein, gel filtration
31200
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calculated from predicted amino acid sequence
38910
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Kar3, molecular mass calculated from the amino acid sequence predicted from the nucleic acid sequence
41600
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ncd, molecular mass calculated from the amino acid sequence predicted from the nucleic acid sequence
46150
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recombinant truncated KAR3, sequence calculation
50839
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2 * 50839, MALDI-TOF MS, the monomer is immobile whereas the fully dimeric KLP-15 construct supports gliding at 0.0023 mm per min and moves towards microtubule minus end
73000
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ncd polypeptide, SDS-PAGE
85000 - 90000
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dynein, SDS-PAGE
87400
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gel filtration
88880
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recombinant Kar3Vik1 heterodimer, sequence calculation
94000
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gel filtration
105000
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ncd-Nkin chimaera, gel filtration
112000
x * 112000, deduced from gene sequence, x * 120000, SDS-PAGE
120000
x * 112000, deduced from gene sequence, x * 120000, SDS-PAGE
182000
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theoretical molecular weight
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 112000, deduced from gene sequence, x * 120000, SDS-PAGE
heterodimer
homodimer
monomer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
dimeric ncd, crystals with the C222(1) space group
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hanging drops,room temperature, PEG 8000
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upon ATP binding, a coiled-coil mechanical element of microtubule-bound Ncd rotates about 79 towards the minus end
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motor domain MgADP complex, hanging drop vapor diffusion method, room temperature, 14% PEG 3350, pH 8.5
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structure of microtubules decorated with Kar3 in the nucleotide-free state, as well as with ADP and 5'-adenylyl-beta,gamma-imidotriphosphate. The empty motor shows large structural changes, including melting of the switch II helix alpha4, closure of nucleotide-binding pocket, and changes in the central beta sheet. Structural changes in switch II control interactions of the Kar3 head with its neck and are transmitted via changes in the central beta sheet of the motor
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80C, 0.3 M NaCl, stored in liquid nitrogen
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
6His-tagged full-length Klp2. a Klp2 protein lacking the motor domain (Klp2-tail) and a Klp2 protein containing only the motor domain and a short coiled coil to mediate dimerization (Klp2-motor) are purified by ammonium sulfate precipitation and HisTrapHP column chromatography
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fusion protein GST-Kar3
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glutathione Sepharose column chromatography
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GST-bind resin chromatography and NAP-5 gel filtration
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His6Kar3/Cik1-S tag protein
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immobilized metal ion affinity chromatography (Ni2+), gel filtration
immobilized metal ion affinity chromatography (Ni2+); recombinant N-terminally His-tagged wild-type and mutant enzymes from Escherichia coli
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ion exchange chromatography (SP Sepharose) and ion exchange chromatography (MonoQ) or gel filtration
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mutated protein molecules carrying point mutations in one of the heads, thus producing heterodimeric motors
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ncd-Nkin chimaera
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partial, recombinant protein, expressed in Escherichia coli
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partially
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
baculovirus constructs encoding the entire CHO2 molecule expressed in Sf9 cells
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claret segregation protein cloned and expressed in bacteria
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DNA sequence analysis of KAR3, Kar3 cloned and expressed in Escherichia coli BL21(DE3)pLysS as fusion protein with glutathione S-transferase, GST-Kar3
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expressed in Escherichia coli BL21 cells
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expressed in Escherichia coli BL21 Star (DE3) or BL21-CodonPlus (DE3) RIL cells
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expressed in Escherichia coli BL21(DE3) pLys strain
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expressed in HeLa cells
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expressed in Sf9 insect cells
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expression in Escherichia coli
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His tagged truncated proteins (e.g. wild type, residue 250-700) expressed in Escherichia coli; recombinant expression of N-terminally His-tagged wild-type and mutant enzymes in Escherichia coli
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His6Kar3/Cik1-S tag protein
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KAR3 gene cloned, Kar3p overexpressed in Escherichia coli
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KLP-15 constructs with different length extensions preceding the motor domain are expressed in Escherichia coli
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motor domain (Leu363-Arg709) expressed in Escherichia coli BL21(DE3)-RIL CodonPlus
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motor domain construct MC6 is expressed in Escherichia coli
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PCR from a ncd cDNA, ncd cloned, polypeptide expressed in Escherichia coli
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pET-ncd, ncd cloned and expressed in Escherichia coli
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pET/MC1 and pET/MC6 encoding dimeric and monomeric ncd expressed in Escherichia coli Bl21(DE3)
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pt7-7, PCR amplification, chimaera of ncd and Neurospora kinesin, cloned and expressed in Escherichia coli Bl21
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recombinant expression of truncated KAR3, coexpression of Kar3 and Vik1 in Escherichia coli strain BL21-CodonPlus(DE)-RIL yielding a Kar3Vik1 heterodimer that forms through the GCN4 leucine zipper
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residues H293-K700 for crystallization, GST fusion protein (residues K210-K700) for biochemical and motility assays expressed in Escherichia coli BL21(DE3)pLysS or Rosetta2(DE3)pLysS
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the gene encoding the enzyme is located on chromosome 16q13-q21, transient overexpression of enzyme KifC3 in COS-7 cells, KifC3 is localized near the nucleus and concentrated at perinuclear microtubule organizing center, overexpression of KifC3 did not affect peroxisomal distribution. Coexpression with human Myc-tagged PEX1
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truncated KAR3 (Lys268-Lys729) and truncated CIK1 (Lys252-Asp594) coexpressed in Escherichia coli BL21-CodonPlus(DE3)-RIL yielding a heterodimer
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truncated proteins and GFP fusion proteins expressed in Sf9 cells and in Escherichia coli BL21-RIL
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E585A
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the mutated amino acids are key residues that are involved in the mediation of structural changes in the head that follow the hydrolysis of the nucelotide. The mutant retains the basal, very low ATPase activity but shows negligible microtubule-stimulated ATPase
E585D
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the mutated amino acids are key residues that are involved in the mediation of structural changes in the head that follow the hydrolysis of the nucelotide. The mutant retains the basal, very low ATPase activity but shows negligible microtubule-stimulated ATPase
G347A
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binds poorly to microtubules in gliding assays, reduced microtubule gliding velocity
G347T
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binds poorly to microtubules in gliding assays, reduced microtubule gliding velocity
M672N
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site-directed mutagenesis, the mutation has a negligible effect on microtubule binding in the absence of nucleotides although substantially changed the Kd(MT) in the presence of AMPPNP
R552A
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the mutated amino acids are key residues that are involved in the mediation of structural changes in the head that follow the hydrolysis of the nucelotide. The mutant retains the basal, very low ATPase activity but shows negligible microtubule-stimulated ATPase
T436S
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the mutant enzyme permits more rapid nucleotide exchange and moves more slowly along microtubles in gliding assays
N593K
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inactive
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
analysis of thermal and urea-induced unfolding of dimeric peptide fragments for identification of coiled coils
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