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Information on EC 2.7.1.160 - 2'-phosphotransferase
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2.7.1.160 2'-phosphotransferaseIUBMB Comments
Catalyses the final step of tRNA splicing in the yeast Saccharomyces cerevisiae . The reaction takes place in two steps: in the first step, the 2'-phosphate on the RNA substrate is ADP-ribosylated, causing the relase of nicotinamide and the formation of the reaction intermediate, ADP-ribosylated tRNA . In the second step, dephosphorylated (mature) tRNA is formed along with ADP ribose 1''-2''-cyclic phosphate. Highly specific for oligonucleotide substrates bearing an internal 2'-phosphate. Oligonucleotides with only a terminal 5'- or 3'-phosphate are not substrates .
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Word Map
- 2.7.1.160
-
2'-phosphate
- junction
- adp-ribose
-
2.7.1.23
- pre-trnas
-
phosphodiester
-
2'-phosphorylated
-
adp-ribosylation
- trinucleotide
-
2'-monophosphate
-
intron-containing
-
aeropyrum
-
phosphoramidite
- trnatyr
-
yeast-like
- pernix
-
anticodon
The expected taxonomic range for this enzyme is: Eukaryota, Archaea, Bacteria
Reaction Schemes
Synonyms
2'-phosphotransferase, AtPt, HeLa cell 2'-phosphotransferase, human TRPT1, KptA, mTPT1, NAD+-dependent 2'-phosphotransferase, NAD-dependent RNA 2'-phosphotransferase, RNA 2'-phosphotransferase, Tpt1, 
more
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
2'-phosphotransferase
KptA
mTPT1
NAD-dependent RNA 2'-phosphotransferase
RNA 2'-phosphotransferase
Tpt1
Tpt1p
tRNA 2'-phosphotransferase
2'-phosphotransferase
-
removes 2'-phosphate from the splice junction of the precursor t-RNA
NAD-dependent RNA 2'-phosphotransferase
Fusobacterium nucleatum subsp. nucleatum ATCC 25586 / JCM14847
-
-
Tpt1p
-
Mammalian genomes encode a single known functional homolog of yeast Tpt1p.
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2'-phospho-[ligated tRNA] + NAD+ = mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
phospho-group transfer
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PATHWAY SOURCE
PATHWAYS
MetaCyc
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SYSTEMATIC NAME
IUBMB Comments
2'-phospho-[ligated tRNA]:NAD+ phosphotransferase
Catalyses the final step of tRNA splicing in the yeast Saccharomyces cerevisiae [2]. The reaction takes place in two steps: in the first step, the 2'-phosphate on the RNA substrate is ADP-ribosylated, causing the relase of nicotinamide and the formation of the reaction intermediate, ADP-ribosylated tRNA [6]. In the second step, dephosphorylated (mature) tRNA is formed along with ADP ribose 1''-2''-cyclic phosphate. Highly specific for oligonucleotide substrates bearing an internal 2'-phosphate. Oligonucleotides with only a terminal 5'- or 3'-phosphate are not substrates [1].
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CAS REGISTRY NUMBER
COMMENTARY
126905-00-8
-
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
18-mer 2' phosphorylated RNA + NAD+
?
-
ACAAGACUGUAA-(2’-phosphate)AUCUUG
-
-
?
2'-phospho-(ligated tRNA) + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide + H+
-
-
-
-
?
2'-phospho-(ligated tRNA) + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide + H2O
yeasts
-
enzyme catalyzes final step in tRNA splicing
-
-
?
2'-phospho-(ligated tRNA) + NAD+
mature tRNA + ADP-ribose1'',2''-phosphate + nicotinamide + H2O
-
last step in the pre-tRNA splicing process
-
-
ir
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP ribose-1'',2''-phosphate + nicotinamide + H2O
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
8-mer 2' phosphorylated RNA + NAD+
?
-
GUAA-(2’-phosphate)AUCU
-
-
?
NAD+ + trinucleotide substrate
ADP-ribosylated RNA
-
requires ca. 0.0004 mM Tpt1 K69A/R71S, only 0.0000003 mM Tpt1p is required for similar or greater product formation
-
-
?
18-mer 2' phosphorylated RNA
?
-
pACAAGACUCUAA-(2’-phosphate)AUCUUG
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP ribose-1'',2''-phosphate + nicotinamide + H2O
-
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP ribose-1'',2''-phosphate + nicotinamide + H2O
-
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP ribose-1'',2''-phosphate + nicotinamide + H2O
-
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP ribose-1'',2''-phosphate + nicotinamide + H2O
-
production of a reaction intermediate, nicotinamide release occurs during intermediate formation, KptA can catalyze the conversion of the intermediate to both product and the original substrate
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP ribose-1'',2''-phosphate + nicotinamide + H2O
-
minimal substrate is a dinucleotide containing an internal 2' phosphate, 600fold more specific for 2'phosphates than for eihter 5'- or 3'-terminal phosphates, 20fold more specific for internal 2' phosphates than for terminal 2' 3', or 2'3'-cyclic phosphates, at least 50fold more specific for internal 2' phosphates than terminal 2' phosphates
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP ribose-1'',2''-phosphate + nicotinamide + H2O
-
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP ribose-1'',2''-phosphate + nicotinamide + H2O
-
mTpt1 can catalyze the conversion of the KptA-generated intermediate to both product and the original substrate
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP ribose-1'',2''-phosphate + nicotinamide + H2O
-
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP ribose-1'',2''-phosphate + nicotinamide + H2O
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP ribose-1'',2''-phosphate + nicotinamide + H2O
-
minimal substrate is a dinucleotide containing an internal 2'-phosphate
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP ribose-1'',2''-phosphate + nicotinamide + H2O
-
specifity for substrates bearing an internal 2'-phosphate
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP ribose-1'',2''-phosphate + nicotinamide + H2O
-
Tpt1 can catalyze the conversion of the KptA-generated intermediate to both product and the original substrate
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP ribose-1'',2''-phosphate + nicotinamide + H2O
-
tRNA_Leu_UAG, tRNA_SER, tRNA_LEU_CAA, tRNA_TYR, tRNA_LYS, tRNA_PRO1, tRNA_PRO2, tRNA_PHE, unidentified tRNA
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP ribose-1'',2''-phosphate + nicotinamide + H2O
-
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
Agrobacterium tumefaciens C58 / ATCC 33970
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
Fusobacterium nucleatum subsp. nucleatum ATCC 25586 / JCM14847
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
3-mer 2' phosphorylated RNA
?
-
pApa-(2’-phosphate)pA, pUpU-(2’-phosphate)pU, pAAA-(2’-phosphate)p and pAAA-(2’-phosphate)pOCH3
-
-
?
3-mer 2' phosphorylated RNA
?
-
pApA-(2’-phosphate)pA, pApU-(2’-phosphate)pA, pUpU-(2’-phosphate)pU, pApApA-(2’-phosphate),pApApA-(2’-phosphate)p, pApApA-(2’-phosphate)1OCH3
-
-
?
additional information
?
-
-
Comparison of incorporation of 3H-tyrosine and 35S-methionine/cysteine into total proteins confirms that wild-type and Trpt1-/-cells have similar incorporation rates of either amino acid.
-
-
?
additional information
?
-
yeasts
-
synthesis of 5'-Lv-2'-DMT-3'-phosphoramidite-G monomer containing an internal 2-phosphate for incorporation in synthetic RNA in order to construct a fluorescent oligoribonucleotide probe for a high throughput screening assay against yeast Tpt1p.
-
-
?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2'-phospho-(ligated tRNA) + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide + H+
-
-
-
-
?
2'-phospho-(ligated tRNA) + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide + H2O
yeasts
-
enzyme catalyzes final step in tRNA splicing
-
-
?
2'-phospho-(ligated tRNA) + NAD+
mature tRNA + ADP-ribose1'',2''-phosphate + nicotinamide + H2O
-
last step in the pre-tRNA splicing process
-
-
ir
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
additional information
?
-
-
Comparison of incorporation of 3H-tyrosine and 35S-methionine/cysteine into total proteins confirms that wild-type and Trpt1-/-cells have similar incorporation rates of either amino acid.
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
Agrobacterium tumefaciens C58 / ATCC 33970
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
Fusobacterium nucleatum subsp. nucleatum ATCC 25586 / JCM14847
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
-
-
-
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
-
UMP, SAMP, cAMP, UTP, CTP, GTP, ATP and derivatives of NAD+ like NMN, ADP-ribose, ATP-ribose, NADPH, NADP+, NADH do not support dephosphorylation
-
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
ApGpApUpUpUpApC
-
oligonucleotide, which lacks secondary structure and has no terminal phosphate
[pre-tRNA]
-
intron-containing yeast [pre-tRNA], which has the same structure as mature yeast [tRNA], except in the region of the intron
-
[tRNA]
-
mature Escherichia coli [tRNA], has canonical tRNA structue
-
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0000017
18-mer 2' phosphorylated RNA
-
pACAAGACUCUAA-(2’-phosphate)AUCUUG, 30°C
-
0.000325
3-mer 2' phosphorylated RNA
-
pUpU-(2’-phosphate)pUs, 30°C
-
0.00065
3-mer 2' phosphorylated RNA
-
pApApA-(2’-phosphate)p, 30°C
-
0.003
3-mer 2' phosphorylated RNA
-
pApApA-(2’-phosphate)pOCH3, 30°C
-
0.00002
8-mer 2' phosphorylated RNA
-
pGUAA-(2’-phosphate)AUCU, 30°C
-
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.04
18-mer 2' phosphorylated RNA
-
pACAAGACUCUAA-(2’-phosphate)AUCUUG, 30°C
-
0.317
8-mer 2' phosphorylated RNA
-
pGUAA-(2’-phosphate)AUCU, 30°C
-
1.173
3-mer 2' phosphorylated RNA
-
pApApA-(2’-phosphate)pOCH3, 30°C
-
6.08
3-mer 2' phosphorylated RNA
-
pApApA-(2’-phosphate)pOCH3, 30°C
-
additional information
18-mer 2' phosphorylated RNA
-
37°C, vmax = 99 fmol/min/ng
-
additional information
3-mer 2' phosphorylated RNA
-
pAAA-(2’-phosphate)p, 37°C, vmax = 1.4 fmol/min/ng
-
additional information
3-mer 2' phosphorylated RNA
-
pAAA-(2’-phosphate)pOCH3, 37°C, vmax = 45 fmol/min/ng
-
additional information
3-mer 2' phosphorylated RNA
-
pApA-(2’-phosphate)pA, 37°C, vmax = 280 fmol/min/ng
-
additional information
3-mer 2' phosphorylated RNA
-
pUpU-(2’-phosphate)pU, 37°C, vmax = 1030 fmol/min/ng
-
additional information
8-mer 2' phosphorylated RNA
-
37°C, vmax = 170 fmol/min/ng
-
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
-
Despite lack of detectable in vitro RNA 2'-phosphotransferase activity in lysates of cells derived from the Trpt1 knockout mice, there is no evidence for a defect in the unfolded protein response or in tRNA function.
additional information
-
Immunoprecipitation of Trpt1 from heart lysates, followed by immunoblot with an antiserum raised to bacterially expressed murine Trpt1 reveals that Trpt1-/-mice lack 2'-phosphotransferase activity yet retain the ability to translate spliced transcription factor XBP-1 mRNA and synthesize tyrosine-containing proteins at wild-type levels. Heterozygotes express 50% of wild-type levels. Extracts of Trpt1+/+cells have measurable NAD+-dependent 2'-phosphotransferase activity.
additional information
-
200000000 units/mg for recombinant Tpt1 produced in Escherichia coli and 140000000 units/mg for Tpt1 purified from yeast
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30
37
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
18 - 30
-
F27A, R71Q, TPT1(1-220) and TPT1(1-210) cells failed to grow at 37°C on yeast medium/peptone/dextrose agar, R71Q and TPT1(1-210) strains grew slower than wild-type TPT1
18 - 37
-
growth of mutants K16A, K69A, H90A, S91A, H117A, T119A, H142A, R158A and R138K on yeast medium/peptone/dextrose agar, colony sizes were similar to that of wild-type TPT1 cells
25 - 30
-
growth of mutants K16A, Y38A, K69A, F72A, H90A, S91A, H117A, T119A, H142A, R158A, TPT1(1-220) and TPT1(1-210) on 5-fluoroorotic acid agar medium
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
plant splicing enzymes fused to GFP by their transient expression in Allium epidermal and Vicia guard cells.
-
-
brenda
-
-
-
brenda
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
-
Clones from two individually selected heterozygous ES cell lines are injected into blastocysts and the resultant founder chimeras bred to obtain two lines of knockout Trpt1+/- and knockout Trpt1-/-mice.
brenda
-
Upper epidermis of Allium cepa is used for transient expression
brenda
-
Lower leaf epidermis from Vicia faba is used for transient expression
brenda
-
Trpt1+/+ and Trpt1-/-cell lines are obtained by serial passage of SV40-T-antigen transfected mouse embryonic fibroblasts
brenda
additional information
additional information
-
nothing detectable in spleen, small intestine, lung, peripheral blood leukocytes, brain and colon
brenda
additional information
nothing detectable in spleen, small intestine, lung, peripheral blood leukocytes, brain and colon
brenda
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
enzyme contains chloroplast transit signals at the N-terminus, 2'-phosphotransferase are not required for classical tRNA splicing in chloroplast, it has an alternative function
brenda
-
2'-phosphotransferase shows a tendency to associate with mitochondria
brenda
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
physiological function
in bacteria NAD-dependent RNA 2'-phosphotransferase KptA might be an RNA processing enzyme able to repair the RNA damaged by toxic endoribonucleases
physiological function
-
in yeast the NAD-dependent RNA 2'-phosphotransferase KptA is known to participate in tRNA splicing by removing the splice junction 2'-phosphate from ligated tRNA
physiological function
the genomic organization of gene kptA suggests that KptA activity is strictly coordinated with the recycling to NAD of its reaction products
Show AA Sequence and Transmembrane Helices (2855 entries)
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
27800
estimated theorethical molecular mass of the deduced translation product
30000
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
hanging-drop vapor-diffusion method, multiwavelength anomalous diffraction method at 2.8 angstrom, structure of enzyme of two globular domains, N and C-domains, which form a cleft in the center
-
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
H117A
H142A
H90A
K16A
-
colony size indistinguishable from that of wild-type TPT1 strain
K69A
-
colony size indistinguishable from that of wild-type TPT1 strain
K69A/R71S
-
this mutant produces a substantial amount of the reaction intermediate
R138A
R138K
-
colony size indistinguishable from that of wild-type TPT1 strain
R158A
-
colony size indistinguishable from that of wild-type TPT1 strain
S91A
T119A
-
colony size indistinguishable from that of wild-type TPT1 strain
Y38A
-
colony size indistinguishable from that of wild-type TPT1 strain
H117A
-
colony size indistinguishable from that of wild-type TPT1 strain
H142A
-
colony size indistinguishable from that of wild-type TPT1 strain
H90A
-
colony size indistinguishable from that of wild-type TPT1 strain
S91A
-
colony size indistinguishable from that of wild-type TPT1 strain
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, Tris buffer, pH 7.5, 50% glycerol
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
by Blue Sepharose column chromatography
immobilized metal ion affinity chromatography, > 80% purified by SDS-PAGE
-
KptA-His6 10% purified by sonication, immobilized metal ion chromatography, centrifugation
-
Ni-agarose chromatography
partially purified
partially purified, GST-Tpt1 fusion protein purified by glutahthione agarose chromatography
-
purified 28000fold by Orange A Sepharose column, 4fold less pure by streamlined purification procedure
Tpt1-His6 50% purified by sonication, immobilized metal ion chromatography, centrifugation
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
expression in Escherichia coli as a His-6 fusion, expression in Saccharomyces cerevisiae tpt1 mutant
expression in Saccharomyces cerevisiae tpt1 mutant strain Y2, the human TRPT1 gene could complement this mutation
expression in Saccharomyces cerevisiae tpt1 mutant strain Y2, the wild-type yeast TPT1 could complement this mutation
-
expression in Saccharomyces cerevisiae tpt1 mutants
Fusion of the coding region of the splicing enzyme in-frame to the amino-terminus of the green fluorescent protein (GFP). Full-length 2'-phosphotransferase cDNA (289 aa) and a short version of the same polypeptide starting with the second methionine at position 33 are fused to the coding region of GFP. Additionally, the N-terminal sequence of 97 amino acids derived from the 2'-phosphotransferase is directly fused to GFP.
-
gene kptA, encoded in an operon-like cluster with genes encoding RNA polymerase, RNA ligase, and the Hen1/Pnkp RNA repairsystem reported to repair ribotoxin-degraded tRNAs in vitro
gene kptA, genomic organization, comparisons
gene kptA, genomic organization, comparisons, the gene kpta neighborhood includes the genes coding for: (i) the enzyme Nam phosphoribo-syltransferase (NadV), involved in the recycling to NAD of Nam, thepyridine product of the KptA-catalyzed reaction, (ii) the orthologue of the yeast ADPR-1-P phosphatase, implicated in the conversionto ADPR of the adenylylic product, (iii) the enzyme ADPR diphosphatase (ADPRP), responsible for ADPR hydrolysis to AMPand R5P, and (iv) the enzyme PRPP synthetase, which catalyzes PRPP formation from ATP and ribose 5-phosphate
The murine Trpt1 gene is targeted in E14 ES cells with a positive/negative selection vector. Trpt1 gene is disrupted by homologous recombination in ES cells, deleting exons 3, 4, and part of exon 5 that encode amino acids 48-173. Clones from two individually selected heterozygous ES cell lines are injected into blastocysts and the resultant founder chimeras bred to obtain two lines of Trpt1+/- and Trpt1-/-mice that are both viable and healthy and are normally fertile. Cell culture: Trpt1+/+ and Trpt1-/-cell lines are obtained by serial passage of SV40-T-antigen transfected mouse embryonic fibroblasts.
-
expression in Escherichia coli as a His-6 fusion, expression in Saccharomyces cerevisiae tpt1 mutant
-
expression in Escherichia coli as a His-6 fusion, expression in Saccharomyces cerevisiae tpt1 mutant
-
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
Inhibitors of Tpt1p might prove useful as selective antifungal agents.
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Culver, G.M.; McCraith, S.M.; Consaul, S.A.; Stanford, D.R.; Phizicky, E.M.
A 2'-phosphotransferase implicated in tRNA splicing is essential in Saccharomyces cerevisiae
J. Biol. Chem.
272
13203-13210
1997
Saccharomyces cerevisiae, Saccharomyces cerevisiae (Q12272), Escherichia coli, Homo sapiens, Mus musculus, Schizosaccharomyces pombe
brenda
Steiger, M.A.; Kierzek, R.; Turner, D.H.; Phizicky, E.M.
Substrate recognition by a yeast 2'-phosphotransferase involved in tRNA splicing and by its Escherichia coli homolog
Biochemistry
40
14098-14105
2001
Saccharomyces cerevisiae, Escherichia coli
brenda
Hu, Q.D.; Lu, H.; Huo, K.; Ying, K.; Li, J.; Xie, Y.; Mao, Y.; Li, Y.Y.
A human homolog of the yeast gene encoding tRNA 2'-phosphotransferase: cloning, characterization and complementation analysis
Cell. Mol. Life Sci.
60
1725-1732
2003
Arabidopsis thaliana, Archaeoglobus fulgidus, Saccharomyces cerevisiae, Clostridium perfringens, Escherichia coli, Fusobacterium nucleatum, Homo sapiens, Homo sapiens (Q86TN4), Methanopyrus kandleri, Mus musculus, Rattus norvegicus, Schizosaccharomyces pombe, Streptomyces coelicolor
brenda
Mc Craith, S.M.; Phizicky, E.M.
An enzyme from Saccharomyces cerevisiae uses NAD+ to transfer the splice junction 2'-phosphate from ligated tRNA to an acceptor molecule
J. Biol. Chem.
266
11986-11992
1991
Saccharomyces cerevisiae
brenda
Zillman, M.; Gorovsky, M.A.; Phizicky, E.M.
HeLa cells contain a 2'-phosphate-specific phosphotransferase similar to a yeast enzyme implicated in tRNA splicing
J. Biol. Chem.
267
10289-10294
1992
Saccharomyces cerevisiae, Homo sapiens
brenda
Spinelli, S.L.; Kierzek, R.; Turner, D.H.; Phizicky, E.M.
Transient ADP-ribosylation of a 2'-phosphate implicated in its removal from ligated tRNA during splicing in yeast
J. Biol. Chem.
274
2637-2644
1999
Saccharomyces cerevisiae, Escherichia coli, Mus musculus
brenda
Kato-Murayama, M.; Bessho, Y.; Shirouzu, M.; Yokoyama, S.
Crystal structure of the RNA 2'-phosphotransferase from Aeropyrum pernix K1
J. Mol. Biol.
348
295-305
2005
Aeropyrum pernix, Arabidopsis thaliana, Archaeoglobus fulgidus, Saccharomyces cerevisiae, Escherichia coli, Homo sapiens, Methanosarcina mazei, Pseudomonas aeruginosa, Pyrococcus horikoshii
brenda
Kierzek, R.; Steiger, M.A.; Spinelli, S.L.; Turner, D.H.; Phizicky, E.M.
The chemical synthesis of oligoribonucleotides with selectively placed 2'-O-phosphates
Nucleosides Nucleotides Nucleic Acids
19
917-933
2000
Saccharomyces cerevisiae
brenda
Spinelli, S.L.; Malik, H.S.; Consaul, S.A.; Phizicky, E.M.
A functional homolog of a yeast tRNA splicing enzyme is conserved in higher eukaryotes and in Escherichia coli
Proc. Natl. Acad. Sci. USA
95
14136-14141
1998
Arabidopsis thaliana, Archaeoglobus fulgidus, Saccharomyces cerevisiae, Candida albicans, Escherichia coli, Mus musculus, no activity in Bacillus subtilis, no activity in Haemophilus influenzae, no activity in Mycoplasma genitalium, Pseudomonas aeruginosa, Pyrococcus horikoshii, Schizosaccharomyces pombe, no activity in Helicobacter pylori
brenda
Sawaya, R.; Schwer, B.; Shuman, S.
Structure-function analysis of the yeast NAD+-dependent tRNA 2'-phosphotransferase Tpt1
RNA
11
107-113
2005
Aeropyrum pernix, Archaeoglobus fulgidus, Saccharomyces cerevisiae, Clostridium perfringens, Drosophila melanogaster, Escherichia coli, Homo sapiens, Leishmania major, Nostoc punctiforme, Pyrococcus horikoshii, Schizosaccharomyces pombe, Trypanosoma cruzi
brenda
Steiger, M.A.; Jackman, J.E.; Phizicky, E.M.
Analysis of 2'-phosphotransferase (Tpt1p) from Saccharomyces cerevisiae: evidence for a conserved two-step reaction mechanism
RNA
11
99-106
2005
Saccharomyces cerevisiae
brenda
Spinelli, S.L.; Consaul, S.A.; Phizicky, E.M.
A conditional lethal yeast phosphotransferase (tpt1) mutant accumulates tRNAs with a 2'-phosphate and an undermodified base at the splice junction
RNA
3
1388-1400
1997
Saccharomyces cerevisiae, Escherichia coli
brenda
Englert, M.; Latz, A.; Becker, D.; Gimple, O.; Beier, H.; Akama, K.
Plant pre-tRNA splicing enzymes are targeted to multiple cellular compartments
Biochimie
89
1351-1365
2007
Arabidopsis thaliana
brenda
Lackey, J.G.; Ron, D.; Damha, M.J.; Harding, H.P.
Toward the discovery of new antifungal agents: the design and validation of a novel 2P-RNA probe and high throughput screening assay against 2-phosphotransferase Tpt1p
Nucleic Acids Symp. Ser.
52
475-476
2008
yeasts
brenda
Harding, H.P.; Lackey, J.G.; Hsu, H.C.; Zhang, Y.; Deng, J.; Xu, R.M.; Damha, M.J.; Ron, D.
An intact unfolded protein response in Trpt1 knockout mice reveals phylogenic divergence in pathways for RNA ligation
RNA
14
225-232
2008
Mus musculus
brenda
Sorci, L.; Ruggieri, S.; Raffaelli, N.
NAD homeostasis in the bacterial response to DNA/RNA damage
DNA Repair
23
17-26
2014
Saccharomyces cerevisiae, Brachyspira hyodysenteriae (A0A3B6VB17), Flavobacterium johnsoniae (A5FLZ4), Herpetosiphon aurantiacus (A9B356), Delftia acidovorans (A9C0S5), Deinococcus deserti (C1CZ01), Catenulispora acidiphila (C7QAQ2), Sebaldella termitidis (D1APE3), Deinococcus gobiensis (H8GTD5), Fusobacterium nucleatum subsp. nucleatum (Q8R5N7), Agrobacterium tumefaciens (Q8U9Z2), Deinococcus radiodurans (Q9RRR1), Delftia acidovorans DSM 14801 (A9C0S5), Agrobacterium tumefaciens C58 / ATCC 33970 (Q8U9Z2), Brachyspira hyodysenteriae ATCC 49526 (A0A3B6VB17), Herpetosiphon aurantiacus DSM 785 (A9B356), Deinococcus gobiensis DSM 21396 (H8GTD5), Deinococcus radiodurans ATCC 13939 (Q9RRR1), Fusobacterium nucleatum subsp. nucleatum ATCC 25586 / JCM14847 (Q8R5N7), Deinococcus deserti DSM 17065 (C1CZ01), Sebaldella termitidis ATCC 33386 (D1APE3)
brenda
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