Information on EC 2.7.1.160 - 2'-phosphotransferase

AtPt

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HeLa cell 2'-phosphotransferase

-

human TRPT1

-

KptA

mTPT1

NAD+-dependent 2'-phosphotransferase

-

NAD-dependent RNA 2'-phosphotransferase

21 entries

RNA 2'-phosphotransferase

29 entries

Tpt1

5 entries

Tpt1p

tRNA 2'-phosphotransferase

12 entries

Trpt1

-

gene

yeast 2'-phosphotransferase

-

667569, 669180, 670431

2'-phospho-[ligated tRNA] + NAD+ = mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide

-

phospho-group transfer

MetaCyc

tRNA splicing I

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].

126905-00-8

-

18-mer 2' phosphorylated RNA

?

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

689261

-

?

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]

?

2'-phospho-[ligated tRNA] + NAD+

mature tRNA + ADP ribose-1'',2''-phosphate + nicotinamide + H2O

14 entries

2'-phospho-[ligated tRNA] + NAD+

mature tRNA + ADP-ribose 1",2"-phosphate + nicotinamide

21 entries

3-mer 2' phosphorylated RNA

?

3-mer 2' phosphorylated RNA + NAD+

?

-

AU-(2’-phosphate)U

-

?

8-mer 2' phosphorylated RNA

?

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

-

?

additional information

?

-

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

689261

-

?

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

21 entries

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.

-

NAD+

16 entries

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

669179

-

Mg2+

additional information

-

Ca2+ and Zn2+ have no effect on activity

669179

ApGpApUpUpUpApC

-

oligonucleotide, which lacks secondary structure and has no terminal phosphate

EDTA

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inhibition of reaction by molar exess of EDTA over Mg2+

669179

[pre-tRNA]

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intron-containing yeast [pre-tRNA], which has the same structure as mature yeast [tRNA], except in the region of the intron

-

[tRNA]

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mature Escherichia coli [tRNA], has canonical tRNA structue

-

0.0000017 - 0.00015

18-mer 2' phosphorylated RNA

-

0.00000035

2'-phospho-[ligated tRNA]

-

0.00015 - 0.04

3-mer 2' phosphorylated RNA

10 entries

-

0.00002 - 0.00025

8-mer 2' phosphorylated RNA

-

0.000004

[ligated tRNA]

-

-

0.04

18-mer 2' phosphorylated RNA

-

pACAAGACUCUAA-(2’-phosphate)AUCUUG, 30°C

-

0.0016

2'-phospho-[ligated tRNA]

-

0.0057 - 6.08

3-mer 2' phosphorylated RNA

7 entries

-

0.317

8-mer 2' phosphorylated RNA

-

pGUAA-(2’-phosphate)AUCU, 30°C

-

additional information

18-mer 2' phosphorylated RNA

4 entries

-

additional information

7.5

-

assay at

30

37

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

Agrobacterium tumefaciens

gene kptA

Brachyspira hyodysenteriae

Q8U9Z2

UniProt

Agrobacterium tumefaciens C58 / ATCC 33970

gene kptA

UniProt

Archaeoglobus fulgidus

-

668216, 669853, 670715, 670874

-

gene kptA

Brachyspira hyodysenteriae ATCC 49526

C0R0E9

UniProt

gene kptA

C0R0E9

UniProt

Candida albicans

-

-

Catenulispora acidiphila

gene kptA

C7QAQ2

UniProt

Clostridium perfringens

-

668216, 670874

-

Deinococcus deserti

gene kptA

Deinococcus deserti DSM 17065

C1CZ01

UniProt

gene kptA

C1CZ01

UniProt

Deinococcus gobiensis

gene kptA

Deinococcus gobiensis DSM 21396

H8GTD5

UniProt

gene kptA

H8GTD5

UniProt

Deinococcus radiodurans

gene kptA

Deinococcus radiodurans ATCC 13939

Q9RRR1

UniProt

gene kptA

UniProt

gene kptA

UniProt

Delftia acidovorans DSM 14801

gene kptA

A9C0S5

UniProt

Drosophila melanogaster

-

-

Flavobacterium johnsoniae

gene kptA

A5FLZ4

UniProt

Fusobacterium nucleatum

-

Fusobacterium nucleatum subsp. nucleatum

-

gene kptA

Q8R5N7

UniProt

Fusobacterium nucleatum subsp. nucleatum ATCC 25586 / JCM14847

gene kptA

Herpetosiphon aurantiacus

Q8R5N7

UniProt

gene kptA

Herpetosiphon aurantiacus DSM 785

A9B356

UniProt

gene kptA

UniProt

-

-

Methanopyrus kandleri

-

-

-

-

no activity in Bacillus subtilis

-

-

no activity in Haemophilus influenzae

-

-

no activity in Helicobacter pylori

-

-

no activity in Mycoplasma genitalium

-

-

Nostoc punctiforme

-

-

Pseudomonas aeruginosa

-

669853, 670715

-

Pyrococcus horikoshii

-

669853, 670715, 670874

-

Rattus norvegicus

-

-

Schizosaccharomyces pombe

Sebaldella termitidis

gene kptA

Sebaldella termitidis ATCC 33386

D1APE3

UniProt

gene kptA

D1APE3

UniProt

Streptomyces coelicolor

-

-

Trypanosoma cruzi

-

-

yeasts

-

689261

-

blastocyst

-

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.

epidermis

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Upper epidermis of Allium cepa is used for transient expression

-

intermediate level of TRPT1 transcript

leaf epidermis

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Lower leaf epidermis from Vicia faba is used for transient expression

intermediate level of TRPT1 transcript

MEF cell

-

Trpt1+/+ and Trpt1-/-cell lines are obtained by serial passage of SV40-T-antigen transfected mouse embryonic fibroblasts

low level of TRPT1 transcript

highest level of TRPT1 transcript

low level of TRPT1 transcript

additional information

chloroplast

-

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

at 11q13

-

-

2'-phosphotransferase shows a tendency to associate with mitochondria

-

-

physiological function

25000

-

gel filtration

27800

Q86TN4

estimated theorethical molecular mass of the deduced translation product

30000

monomer

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

-

669853

H22A

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lethal in vivo

R125A

-

lethal in vivo

R21A

-

lethal in vivo

R69A

-

lethal in vivo

C85R

-

no accumulation of the reaction intermediate

D34A

-

no accumulation of the reaction intermediate

F72A

-

temperature sensitive

H117A

H117A-T119A

-

lethal in vivo

H140A

-

no accumulation of the reaction intermediate

H140A/H142A

-

no accumulation of the reaction intermediate

H140A/V141A/H142A

-

no accumulation of the reaction intermediate

H142A

H24A

-

lethal in vivo

H24N

-

lethal in vivo

H24Q

-

lethal in vivo

H90A

I167L/D168K

-

no accumulation of the reaction intermediate

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

M136G

-

no accumulation of the reaction intermediate

N185A

-

no accumulation of the reaction intermediate

Q88A/H90A/S91A

-

no accumulation of the reaction intermediate

Q88Y

-

no accumulation of the reaction intermediate

R138A

R138K

-

colony size indistinguishable from that of wild-type TPT1 strain

R138Q

-

lethal in vivo

R158A

-

colony size indistinguishable from that of wild-type TPT1 strain

R158H

-

no accumulation of the reaction intermediate

R23A

-

lethal in vivo

R23A/H24A

-

no accumulation of the reaction intermediate

R23K

-

lethal in vivo

R23Q

-

lethal in vivo

R71A

-

lethal in vivo

R71K

-

lethal in vivo

R71Q

-

grows slower than wild-type, temperature sensitive

S155A

-

no accumulation of the reaction intermediate

S15A/K16A

-

no accumulation of the reaction intermediate

S183A

-

no accumulation of the reaction intermediate

S91A

T119A

-

colony size indistinguishable from that of wild-type TPT1 strain

Y38A

-

colony size indistinguishable from that of wild-type TPT1 strain

-20, Tris-HCl buffer, pH. 8.0, 50% glycerol, several months

-

669180

-20°C, Tris buffer, pH 7.5, 50% glycerol

-20°C, Tris-buffer, pH 7.5, 50% (m/v) glycerol, 3 months

Q12272

662096

-80, Tris-HCl buffer, pH 8.0, 10% glycerol

-

-80°C, Tris-HCl buffer, pH 8.0, 20% glycerol

-

669180

by Blue Sepharose column chromatography

by nickel affinity chromatography

-

669189

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 as a His6-tagged protein

-

partially purified by Blue sepharose column chromatography

-

670876

partially purified by chromatography on heparin-agarose

-

669180

partially purified, GST-Tpt1 fusion protein purified by glutahthione agarose chromatography

-

670876

purified 28000fold by Orange A Sepharose column, 4fold less pure by streamlined purification procedure

Q12272

662096

sonication, centrifugation

-

669853

Tpt1-His6 50% purified by sonication, immobilized metal ion chromatography, centrifugation

-

expression from Saccharomyces cerevisiae vectors

-

expression in Escherichia coli

4 entries

expression in Escherichia coli as a His-6 fusion

-

expression in Escherichia coli as a His-6 fusion, expression in Saccharomyces cerevisiae tpt1 mutant

expression in Escherichia coli B834(DE3)

-

669853

expression in Escherichia coli BL21(DE3)

-

expression in Escherichia coli strain RZ510

Q12272

662096

expression in Saccharomyces cerevisiae

-

expression in Saccharomyces cerevisiae tpt1 mutant strain Y2, the human TRPT1 gene could complement this mutation

Q86TN4

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

Catenulispora acidiphila

C7QAQ2

Flavobacterium johnsoniae

gene kptA, genomic organization, comparisons

9 entries

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

A5FLZ4

overexpression in Escherichia coli BL21(DE3)

-

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.

-