Information on EC 6.3.2.25 - Tubulin-tyrosine ligase

New: Word Map on EC 6.3.2.25
Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
Specify your search results
Mark a special word or phrase in this record:
Search Reference ID:
Select one or more organisms in this record:
Show additional data
Do not include text mining results
Include (text mining) results (more...)
Include results (AMENDA + additional results, but less precise; more...)


The expected taxonomic range for this enzyme is: Eukaryota

EC NUMBER
COMMENTARY hide
6.3.2.25
-
RECOMMENDED NAME
GeneOntology No.
Tubulin-tyrosine ligase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + detyrosinated alpha-tubulin + L-tyrosine = alpha-tubulin + ADP + phosphate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
formation of peptide bond
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
alpha-Tubulin:L-tyrosine ligase (ADP-forming)
L-Tyrosine is linked via a peptide bond to the C-terminus of de-tyrosinated alpha-tubulin (des-Tyromega-alpha-tubulin). The enzyme is highly specific for alpha-tubulin and moderately specific for ATP and L-tyrosine. L-Phenylalanine and 3,4-dihydroxy-L-phenylalanine are transferred but with higher Km values.
CAS REGISTRY NUMBER
COMMENTARY hide
60321-03-1
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
cv. Bright Yellow 2, cell culture BY-2
-
-
Manually annotated by BRENDA team
cv. Nihonmasari
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
-
mutation of tubulin binding residues largely abolishes the enzyme ability to tyrosinate alpha-tubulin and restrict neurite outgrowth in cultured neurons. The neuronal defects of tubulin tyrosine ligase knockout mice are due to the loss of tubulin tyrosination and not because the enzyme is required to tyrosinate any other substrates. Neurons from TTL-null mice show strong developmental defects, including increased neurite extensions and premature differentiation
physiological function
additional information
the enzyme's tubulin-contacting residues are well conserved. The alpha-tubulin residues alphaGlu441 and alphaGlu449 anchor the tail by forming hydrogen bonds with residues Arg73, Ala75, Ser76, Ser152, and Val179, and Asn10, Ser12, Arg44, and Pro336 of TTL, respectively. When bound to the enzyme, the polypeptide chain of the alpha-tubulin tail adopts a loop-like conformation between the tail-anchoring residues alphaGlu441 and alphaGlu449. The enzyme's orientation on tubulin heterodimers places its catalytic domain near to alpha-tubulin's C-terminal tail, which binds to the enzyme's active site through two glutamate residues missing from beta-tubulin's C-terminus
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + detyrosinated alpha-tubulin + 3-fluoro-Tyr
?
show the reaction diagram
-
tubulin covalently labeled with 3-fluoro-Tyr is competient to polimerize into microtubules
-
-
-
ATP + detyrosinated alpha-tubulin + 3-nitro-L-tyrosine
nitrotyrosinated alpha-tubulin + ADP + phosphate
show the reaction diagram
ATP + detyrosinated alpha-tubulin + iodotyrosine
?
show the reaction diagram
-
-
-
-
-
ATP + detyrosinated alpha-tubulin + L-DOPA
?
show the reaction diagram
-
-
-
-
-
ATP + detyrosinated alpha-tubulin + L-hydroxyphenylalanine
?
show the reaction diagram
-
-
-
-
-
ATP + detyrosinated alpha-tubulin + L-Phe
?
show the reaction diagram
ATP + detyrosinated alpha-tubulin + L-Tyr
?
show the reaction diagram
ATP + detyrosinated alpha-tubulin + L-Tyr
alpha-tubulin + ADP + phosphate
show the reaction diagram
ATP + detyrosinated alpha-tubulin + L-tyrosine
alpha-tubulin + ADP + phosphate
show the reaction diagram
ATP + detyrosinated alpha-tubulin + tyramine
?
show the reaction diagram
ATP + peptide of detyrosinated alpha tubulin + Tyr
peptide of alpha-tubulin + ADP + phosphate
show the reaction diagram
-
peptides ending like alpha-tubulin with the sequence Gly-Glu-Glu are optimally tyrosinated once a peptide length of 12 residues is reached. The carboxy-terminal tetradecapeptide of detyrosinated alpha-tubulin acts as substrate at 50fold lower efficiency than alphabeta-tubulin
-
-
-
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
ATP + detyrosinated alpha-tubulin + L-Tyr
?
show the reaction diagram
ATP + detyrosinated alpha-tubulin + L-Tyr
alpha-tubulin + ADP + phosphate
show the reaction diagram
ATP + detyrosinated alpha-tubulin + L-tyrosine
alpha-tubulin + ADP + phosphate
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
-
required for the N-formyl-Met-Leu-Phe-induced and the antibiotic A23187-induced stimulation
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(1R,4aS,6R,8S)-6,8-dihydroxy-5-[(3E)-5-hydroxy-5-(3-hydroxy-5-oxo-2,5-dihydrofuran-2-yl)-3-methylpent-3-en-1-yl]-4a,6-dimethyldecahydronaphthalene-1-carboxylic acid
-
increases the non-tyrosinatable form of alpha-tubulin in human cancer cell cultures indicating to act as an inhibitor of TTL
3-nitro-L-tyrosine
-
50% inhibition of tyrosine incorporation at 50fold higher concentrations of nitrotyrosine
ADP
-
competitive with respect to ATP, non-competitive with respect to tubulin and L-Tyr
alpha-L-Glu-L-Tyr
-
-
AMP
-
-
diiodotyrosine
-
-
L-Ala-L-Tyr
-
-
L-hydroxyphenylalanine
-
-
L-Tyr
-
-
L-Tyr-Gly
-
-
L-Tyr-L-Ala
-
-
L-Tyr-L-Glu
-
-
L-Tyr-L-Tyr
-
-
monoiodotyrosine
-
-
p-Chloromercuriphenyl sulfonic acid
-
-
p-Iodophenylalanine
-
-
phosphate
-
very weak
stathmin
tyramine
-
-
Tyrosinated tubulin
-
very weak
-
additional information
-
no inhibition by thyronine and its iodinated derivatives
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
A23187
-
Ca2+ is required for the N-formyl-Met-Leu-Phe-induced and the A23187-induced stimulation
N-Formyl-Met-Leu-Phe
-
Ca2+ is required for the N-formyl-Met-Leu-Phe-induced and the A23187-induced stimulation
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.014
ATP
-
-
0.186
L-Dopa
-
-
0.16
L-hydroxyphenylalanine
-
-
0.432 - 2.9
L-Phe
0.017 - 0.045
L-Tyr
0.0068
L-tyrosine
-
37°C
0.002
tubulin
-
below 0.002 mM
-
8
tyramine
-
-
0.0019
untyrosinated tubulin
-
-
-
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.24
3-nitro-L-tyrosine
-
37°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.011
-
-
0.039
-
-
0.324
-
-
0.737
-
-
additional information
-
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.51
-
predicted from nucleotide sequence
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
juvenile larval stages L1-L4
Manually annotated by BRENDA team
preferentially expressed in adult and fetal lung
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35000
-
gel filtration, MW of enzyme in crude extract, the MW of the purified enzyme is 150000. Addition of pure dimeric tubulin to the 35000 MW enzyme converts it back to the larger form which is apparently a stoichiometric 1:1 complex of tubulin and the 35000 MW enzyme
37000
-
gel filtration
40000
-
glycerol density gradient centrifugation. The MW of the ligase preincubated with excess phosphocellulose-tubulin is 150000
43000
-
glycerol gradient centrifugation
45000
-
loaded with binding partners, mass spectrometry
additional information
-
two-domain structure: the two domains interact tightly under physiological conditions. The 30000 MW domain carries the binding sites for beta-tubulin and ATP. The 14000 MW domain can possibly form an additional part of the catalytic site
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
-
x * 43000, SDS-PAGE, immunoblot
monomer
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phosphoprotein
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified enzyme in complex with alpha- and beta-tubulin heterodimers, X-ray diffraction structure determination and analysis
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
photoinactivation in presence of 3-azido-L-Tyr and p-azido-L-Phe. PCMB protects by reversibly blocking essential thiol groups during illumination
-
stabilized to dilution by addition of 0.1 mg/ml tubulin
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70°C
-
-70°C, 25 mM K+-morpholinoethanesulfonic acid, pH 6.8, 150 mM KCl, 12.5 MgCl2, 2.5 mM ATP, 1 mM dithiothreitol, 15% glycerol, at least 4 months, no loss of activity
-
-70°C, in presence of 40% or 20% glycerol the enzyme can be stored for at least 6 months at a protein concentration of 0.02 mg/ml
-
-70°C, protein concentration 0.02 mg/ml, 20-40% glycerol, stable for at least 1 year
-
-70°C, stable for at least a year in 400 mM KCl with a protein concentration of about 1 mg/ml
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
GST-TTL fusion protein, glutathione-sepharose affinity chromatography
-
recombinant GST-tagged enzyme from Escherichia coli strain BL21 by glutathione affinity chromatography
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning of cDNA
-
expression in Escherichia coli
-
expression in HEK293T cells
expression of GST-tagged TTL in Escherichia coli strain BL21, co-expression of TTL with MAP1B in COS-7 cells inducing an increase in tyrosinated microtubules
-
expression of TTL in Escherichia coli and Sf9 insect cells
-
transient transfection of a TTL cDNA portion that encodes the beta-tubulin binding domain but lacks the catalytic domain into CHO-K1 cells
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E331Q
site-directed mutagenesis, catalytically inactive mutant
H54A
site-directed mutagenesis, the mutant enzyme shows moderately reduced binding to tubulin dimers
R36E
site-directed mutagenesis, the mutant enzyme shows strongly reduced binding to tubulin dimers
R51A
site-directed mutagenesis, the mutant enzyme shows moderately reduced binding to tubulin dimers
R51A/H54A
site-directed mutagenesis, the mutant enzyme shows strongly reduced binding to tubulin dimers
R66E
site-directed mutagenesis, the mutant enzyme shows strongly reduced binding to tubulin dimers
S152E
site-directed mutagenesis, the mutant shows over 90% reduced activity compared to the wild-type enzyme
S76E
site-directed mutagenesis, the mutant is comparable to the wild-type enzyme
additional information
-
neurons lacking MAP1B, when exposed to drugs that reversibly depolymerize microtubules, do not fully recover tyrosinated microtubules upon drug removal
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
-
utilization of ligase to determine the state of tyrosination of tubulin
synthesis
-
restoration of the excitability of the giant axon of Doryteuthis bleekeri by tubulin-tyrosine ligase and microtubule proteins
Show AA Sequence (144 entries)
Please use the Sequence Search for a certain query.