Information on EC 2.7.7.87 - L-threonylcarbamoyladenylate synthase

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

EC NUMBER
COMMENTARY hide
2.7.7.87
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RECOMMENDED NAME
GeneOntology No.
L-threonylcarbamoyladenylate synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
L-threonine + ATP + HCO3- = L-threonylcarbamoyladenylate + diphosphate + H2O
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
N6-L-threonylcarbamoyladenosine37-modified tRNA biosynthesis
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threonine metabolism
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SYSTEMATIC NAME
IUBMB Comments
ATP:L-threonyl,bicarbonate adenylyltransferase
The enzyme is involved in the synthesis of N6-threonylcarbamoyladenosine37 in tRNAs, with the anticodon NNU, i.e. tRNAIle, tRNAThr, tRNAAsn, tRNALys, tRNASer and tRNAArg [6].
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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the loss of SUA5 causes increased leaky scanning through AUG codons, +1 frameshifting, and nonsense suppression. In addition, the loss of SUA5 amplifies the 20S RNA virus found in Saccharomyces cerevisiae, possibly through an internal ribosome entry site-mediated mechanism
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
L-threonine + ATP + bicarbonate
L-threonylcarbamoyladenylate + diphosphate + H2O
show the reaction diagram
L-threonine + ATP + CO2
L-threonylcarbamoyl-AMP + diphosphate
show the reaction diagram
L-threonylcarbamoyl-AMP + tRNA
t6A37-tRNA
show the reaction diagram
additional information
?
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t6A is a universally conserved tRNA modification found at position 37 in the anticodon loop of a subset of tRNA, structural studies predict an important role for t6A in translational fidelity
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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
L-threonine + ATP + bicarbonate
L-threonylcarbamoyladenylate + diphosphate + H2O
show the reaction diagram
L-threonine + ATP + CO2
L-threonylcarbamoyl-AMP + diphosphate
show the reaction diagram
L-threonylcarbamoyl-AMP + tRNA
t6A37-tRNA
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00011
L-threonine
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pH 8.0, temperature not specified in the publication
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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65% of activity still retained at 73°C
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Pyrococcus abyssi (strain GE5 / Orsay)
Pyrococcus abyssi (strain GE5 / Orsay)
Pyrococcus abyssi (strain GE5 / Orsay)
Sulfurisphaera tokodaii (strain DSM 16993 / JCM 10545 / NBRC 100140 / 7)
Sulfurisphaera tokodaii (strain DSM 16993 / JCM 10545 / NBRC 100140 / 7)
Sulfurisphaera tokodaii (strain DSM 16993 / JCM 10545 / NBRC 100140 / 7)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
38200
determined by analytical ultracentrifugation at 20°C and at 25°C
90500
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theoretical mass of PaKEOPS complex
200000
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recombinant PaKEOPS complex consisting of Kae1, Bud32, Cgi121 and Pcc1, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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2 * 90500
monomer
1 * 38200, analytical ultracentrifugation, Sulfolobus tokodaii Sua5 exists as a monomer in solution
additional information
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fluorescence quenching data indicate that YrdC recognizes the RNA substrate in a sequence-dependent manner by directly interacting with certain bases
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystals grown at 30°C by sitting drop method, complexed with L-threonine and AMPPNP at 1.8 A resolution
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
attempts to purify the native forms of YdiC and YdiE by specific cleavage of the fusion proteins with thrombin or Factor Xa protease lead to multiple products, perhaps because the proteins are not completely stable in the cleavage reaction conditions
by Ni-NTA affinity chromatography to a purity of more than 95% by SDS-PAGE
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gravity-flow chromatography on an Ni-NTA resin column, not associated with tRNAs when purified
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gravity-flow chromatography on an Ni-NTA resin column, PaKEOPS complex co-purified with nucleic acids
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Ni-NTA affinity chromatography
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yrdC protein purified by Ni-NTA affinity chromatography, dialyzed against phosphate-buffered saline and concentrated using a YM-3 Centriprep filter
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a polycistronic sequence containing the genes encoding the PaKEOPS protein subunits Kae, Bud32, Cgi121 and Pcc1 synthesized and cloned into pET28(a) plasmid and transformed in Escherichia coli, Pcc1 fused to a hexa-histidine tag, PaSua5 cloned into pET26b
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Escherichia coli BL21(DE3) cells transformed with a pET21 derivative (Novagen) vector containing the Escherichia coli yrdC gene
full length Sua5 expressed in Escherichia coli BL21 (DE3) CodonPlus cells harboring an N-terminal hexa-histidine tag
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His6-tagged YrdC protein overexpressed in Escherichia coli
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ScSua5 gene amplified by PCR using genomic DNA from Saccharomyces cerevisiae, fused to a hexa-histidine tag at its 3'-end and cloned into pET21d vector and transformed in Escherichia coli
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SDS-PAGE, cloning of ywlC and ydiB as His6 N-fusion proteins and highly expression in Escherichia coli BL21(DE3), because of inclusion bodies construction of N-terminal thioredoxin (trxA) fusion proteins of ydiC and ydiE, which allow good expression and sufficient solubility for in vitro studies
Sulfolobus tokodaii Sua5 protein overexpressed in Escherichia coli
yrdC, yjeE and yeaZ genes from Escherichia coli expressed as N-terminal His6-fusion proteins, YgjD N-terminal as His6-SUMO fusion protein in Escherichia coli
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