Information on EC 2.7.1.160 - 2'-phosphotransferase

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

EC NUMBER
COMMENTARY hide
2.7.1.160
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RECOMMENDED NAME
GeneOntology No.
2'-phosphotransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2'-phospho-[ligated tRNA] + NAD+ = mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide
show the reaction diagram
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-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phospho-group transfer
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
tRNA splicing I
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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].
CAS REGISTRY NUMBER
COMMENTARY hide
126905-00-8
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene kptA
UniProt
Manually annotated by BRENDA team
gene kptA
UniProt
Manually annotated by BRENDA team
gene kptA
UniProt
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
gene kptA
UniProt
Manually annotated by BRENDA team
gene kptA
UniProt
Manually annotated by BRENDA team
gene kptA
UniProt
Manually annotated by BRENDA team
gene kptA
UniProt
Manually annotated by BRENDA team
gene kptA
UniProt
Manually annotated by BRENDA team
gene kptA
UniProt
Manually annotated by BRENDA team
gene kptA
UniProt
Manually annotated by BRENDA team
gene kptA
UniProt
Manually annotated by BRENDA team
K12 strain
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-
Manually annotated by BRENDA team
gene kptA
UniProt
Manually annotated by BRENDA team
gene kptA
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
no activity in Bacillus subtilis
-
-
-
Manually annotated by BRENDA team
no activity in Haemophilus influenzae
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-
-
Manually annotated by BRENDA team
no activity in Helicobacter pylori
-
-
-
Manually annotated by BRENDA team
no activity in Mycoplasma genitalium
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-
-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
gene kptA
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
yeasts
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-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
18-mer 2' phosphorylated RNA
?
show the reaction diagram
18-mer 2' phosphorylated RNA + NAD+
?
show the reaction diagram
-
ACAAGACUGUAA-(2’-phosphate)AUCUUG
-
-
?
2'-phospho-(ligated tRNA) + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide + H+
show the reaction diagram
-
-
-
-
?
2'-phospho-(ligated tRNA) + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide + H2O
show the reaction diagram
yeasts
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enzyme catalyzes final step in tRNA splicing
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-
?
2'-phospho-(ligated tRNA) + NAD+
mature tRNA + ADP-ribose1'',2''-phosphate + nicotinamide + H2O
show the reaction diagram
-
last step in the pre-tRNA splicing process
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-
ir
2'-phospho-[ligated tRNA]
?
show the reaction diagram
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP ribose-1'',2''-phosphate + nicotinamide + H2O
show the reaction diagram
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1",2"-phosphate + nicotinamide
show the reaction diagram
3-mer 2' phosphorylated RNA
?
show the reaction diagram
3-mer 2' phosphorylated RNA + NAD+
?
show the reaction diagram
-
AU-(2’-phosphate)U
-
-
?
8-mer 2' phosphorylated RNA
?
show the reaction diagram
8-mer 2' phosphorylated RNA + NAD+
?
show the reaction diagram
-
GUAA-(2’-phosphate)AUCU
-
-
?
NAD+ + trinucleotide substrate
ADP-ribosylated RNA
show the reaction diagram
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requires ca. 0.0004 mM Tpt1 K69A/R71S, only 0.0000003 mM Tpt1p is required for similar or greater product formation
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-
?
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
2'-phospho-(ligated tRNA) + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide + H+
show the reaction diagram
-
-
-
-
?
2'-phospho-(ligated tRNA) + NAD+
mature tRNA + ADP-ribose 1'',2''-phosphate + nicotinamide + H2O
show the reaction diagram
yeasts
-
enzyme catalyzes final step in tRNA splicing
-
-
?
2'-phospho-(ligated tRNA) + NAD+
mature tRNA + ADP-ribose1'',2''-phosphate + nicotinamide + H2O
show the reaction diagram
-
last step in the pre-tRNA splicing process
-
-
ir
2'-phospho-[ligated tRNA] + NAD+
mature tRNA + ADP-ribose 1",2"-phosphate + nicotinamide
show the reaction diagram
additional information
?
-
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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.
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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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 hide
LITERATURE
additional information
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Ca2+ and Zn2+ have no effect on activity
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ApGpApUpUpUpApC
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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+
[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
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[tRNA]
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mature Escherichia coli [tRNA], has canonical tRNA structue
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0000017 - 0.00015
18-mer 2' phosphorylated RNA
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0.00000035
2'-phospho-[ligated tRNA]
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0.00015 - 0.04
3-mer 2' phosphorylated RNA
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0.00002 - 0.00025
8-mer 2' phosphorylated RNA
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0.000004
[ligated tRNA]
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.04
18-mer 2' phosphorylated RNA
Saccharomyces cerevisiae
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pACAAGACUCUAA-(2’-phosphate)AUCUUG, 30°C
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0.0016
2'-phospho-[ligated tRNA]
Saccharomyces cerevisiae
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0.0057 - 6.08
3-mer 2' phosphorylated RNA
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0.317
8-mer 2' phosphorylated RNA
Saccharomyces cerevisiae
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pGUAA-(2’-phosphate)AUCU, 30°C
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additional information
18-mer 2' phosphorylated RNA
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
18 - 30
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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
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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
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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
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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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.
Manually annotated by BRENDA team
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Upper epidermis of Allium cepa is used for transient expression
Manually annotated by BRENDA team
intermediate level of TRPT1 transcript
Manually annotated by BRENDA team
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Lower leaf epidermis from Vicia faba is used for transient expression
Manually annotated by BRENDA team
intermediate level of TRPT1 transcript
Manually annotated by BRENDA team
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Trpt1+/+ and Trpt1-/-cell lines are obtained by serial passage of SV40-T-antigen transfected mouse embryonic fibroblasts
Manually annotated by BRENDA team
low level of TRPT1 transcript
Manually annotated by BRENDA team
highest level of TRPT1 transcript
Manually annotated by BRENDA team
low level of TRPT1 transcript
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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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
Manually annotated by BRENDA team
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2'-phosphotransferase shows a tendency to associate with mitochondria
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25000
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gel filtration
27800
estimated theorethical molecular mass of the deduced translation product
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
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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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20, Tris-HCl buffer, pH. 8.0, 50% glycerol, several months
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-20°C, Tris buffer, pH 7.5, 50% glycerol
-20°C, Tris-buffer, pH 7.5, 50% (m/v) glycerol, 3 months
-80, Tris-HCl buffer, pH 8.0, 10% glycerol
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-80°C, Tris-HCl buffer, pH 8.0, 20% glycerol
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by Blue Sepharose column chromatography
by nickel affinity chromatography
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immobilized metal ion affinity chromatography, > 80% purified by SDS-PAGE
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KptA-His6 10% purified by sonication, immobilized metal ion chromatography, centrifugation
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Ni-agarose chromatography
partially purified
partially purified as a His6-tagged protein
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partially purified by Blue sepharose column chromatography
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partially purified by chromatography on heparin-agarose
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partially purified, GST-Tpt1 fusion protein purified by glutahthione agarose chromatography
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purified 28000fold by Orange A Sepharose column, 4fold less pure by streamlined purification procedure
sonication, centrifugation
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Tpt1-His6 50% purified by sonication, immobilized metal ion chromatography, centrifugation
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression from Saccharomyces cerevisiae vectors
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expression in Escherichia coli
expression in Escherichia coli as a His-6 fusion
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expression in Escherichia coli as a His-6 fusion, expression in Saccharomyces cerevisiae tpt1 mutant
expression in Escherichia coli B834(DE3)
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expression in Escherichia coli BL21(DE3)
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expression in Escherichia coli strain RZ510
expression in Saccharomyces cerevisiae
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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
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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.
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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
overexpression in Escherichia coli BL21(DE3)
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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.
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H22A
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lethal in vivo
R125A
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lethal in vivo
R21A
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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-T119A
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lethal in vivo
H140A
-
no accumulation of the reaction intermediate
H140A/H142A
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no accumulation of the reaction intermediate
H140A/V141A/H142A
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no accumulation of the reaction intermediate
H24A
-
lethal in vivo
H24N
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lethal in vivo
H24Q
-
lethal in vivo
I167L/D168K
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no accumulation of the reaction intermediate
K16A
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colony size indistinguishable from that of wild-type TPT1 strain
K69A
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colony size indistinguishable from that of wild-type TPT1 strain
K69A/R71S
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this mutant produces a substantial amount of the reaction intermediate
M136G
-
no accumulation of the reaction intermediate
N185A
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no accumulation of the reaction intermediate
Q88A/H90A/S91A
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no accumulation of the reaction intermediate
Q88Y
-
no accumulation of the reaction intermediate
R138K
-
colony size indistinguishable from that of wild-type TPT1 strain
R138Q
-
lethal in vivo
R158A
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colony size indistinguishable from that of wild-type TPT1 strain
R158H
-
no accumulation of the reaction intermediate
R23A
-
lethal in vivo
R23A/H24A
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no accumulation of the reaction intermediate
R23K
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lethal in vivo
R23Q
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lethal in vivo
R71A
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lethal in vivo
R71K
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lethal in vivo
R71Q
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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
T119A
-
colony size indistinguishable from that of wild-type TPT1 strain
Y38A
-
colony size indistinguishable from that of wild-type TPT1 strain
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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Inhibitors of Tpt1p might prove useful as selective antifungal agents.