Information on EC 3.1.1.29 - aminoacyl-tRNA hydrolase

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

EC NUMBER
COMMENTARY hide
3.1.1.29
-
RECOMMENDED NAME
GeneOntology No.
aminoacyl-tRNA hydrolase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
N-substituted aminoacyl-tRNA + H2O = N-substituted amino acid + tRNA
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of carboxylic ester
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
L-asparagine biosynthesis III (tRNA-dependent)
-
-
L-cysteine biosynthesis II (tRNA-dependent)
-
-
SYSTEMATIC NAME
IUBMB Comments
aminoacyl-tRNA aminoacylhydrolase
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CAS REGISTRY NUMBER
COMMENTARY hide
9054-98-2
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
essential gene required for both vegetative growth and sporulation
-
-
Manually annotated by BRENDA team
MRE 600
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Wistar
-
-
Manually annotated by BRENDA team
Wistar
-
-
Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
-
since build-up of peptidyl-tRNAs is toxic, defects in enzyme function result in cell death
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
2'(3')-O-L-(N,N-diacetyl-lysinyl)adenosine + H2O
?
show the reaction diagram
minimalist substrate
-
-
?
acetyl-histidine-tRNA + H2O
acetyl-histidine + tRNA
show the reaction diagram
-
-
-
?
acetyl-histidyl-tRNAHis + H2O
acetyl-histidine + tRNAHis
show the reaction diagram
-
-
-
-
?
Ala-tRNA + H2O
Ala + tRNA
show the reaction diagram
-
-
-
-
?
bulk peptidyl-tRNA + H2O
?
show the reaction diagram
D-tyrosine-tRNA + H2O
D-tyrosine + tRNA
show the reaction diagram
dephosphorylated diacetyl-lysine-tRNA + H2O
dephosphorylated diacetyl-lysine + tRNA
show the reaction diagram
-
-
-
?
dephosporylated formyl-methionine-tRNA + H2O
dephosphorylated formyl-methionine + tRNA
show the reaction diagram
-
-
-
?
diacetyl-Lys-tRNALys + H2O
diacetyl-Lys + tRNA
show the reaction diagram
diacetyl-lysine-tRNA + H2O
diacetyl-lysine + tRNA
show the reaction diagram
diacetyl-lysyl-tRNALys
diacetyl-Lys + tRNALys
show the reaction diagram
-
-
-
-
?
diacetyl-lysyl-tRNALys + H2O
diacetyl-lysine + tRNALys
show the reaction diagram
formyl-Met-tRNAfMet + H2O
formyl-Met + tRNAfMet
show the reaction diagram
-
-
-
-
?
formyl-methionine-tRNA + H2O
formyl-methionine + tRNA
show the reaction diagram
-
-
-
?
formyl-methionyl-tRNAfMet + H2O
formyl-methionine + tRNAfMet
show the reaction diagram
-
Escherichia coli formyl-methionyltRNAfMet, phosphorylated and dephosphorylated substrate
-
-
?
Glu-tRNA + H2O
Glu + tRNA
show the reaction diagram
-
-
-
-
?
Gly-tRNA + H2O
Gly + tRNA
show the reaction diagram
-
-
-
-
?
Gly-tRNAAla + H2O
Gly + tRNAAla
show the reaction diagram
-
-
-
?
L-Lys-tRNALys + H2O
L-lysine + tRNALys
show the reaction diagram
-
-
-
?
Leu-tRNA + H2O
Leu + tRNA
show the reaction diagram
Lys-tRNA + H2O
Lys + tRNA
show the reaction diagram
Met-tRNA + H2O
Met + tRNA
show the reaction diagram
-
-
-
-
?
Met-tRNAMet + H2O
Met + tRNAMet
show the reaction diagram
-
-
-
-
?
N-acetyl-Ala-tRNA + H2O
N-acetyl-Ala + tRNA
show the reaction diagram
-
-
-
-
?
N-acetyl-Ala-tRNA(Ala) + H2O
N-acetyl-Ala + tRNA(Ala)
show the reaction diagram
-
-
-
-
?
N-acetyl-Glu-tRNA + H2O
N-acetyl-Glu + tRNA
show the reaction diagram
-
-
-
-
?
N-acetyl-His-tRNA + H2O
N-acetyl-His + tRNA
show the reaction diagram
-
-
-
-
?
N-acetyl-Leu-Gly-tRNA + H2O
N-acetyl-Leu-Gly + tRNA
show the reaction diagram
-
-
-
-
?
N-acetyl-Leu-tRNA + H2O
N-acetyl-Leu + tRNA
show the reaction diagram
N-acetyl-Lys-tRNA + H2O
N-acetyl-Lys + tRNA
show the reaction diagram
N-acetyl-Met-tRNA + H2O
N-acetyl-Met + tRNA
show the reaction diagram
-
-
-
-
?
N-acetyl-Phe-Phe-tRNA + H2O
N-acetyl-Phe-Phe + tRNA
show the reaction diagram
-
-
-
-
?
N-acetyl-Phe-tRNA + H2O
N-acetyl-L-Phe + tRNA
show the reaction diagram
-
-
-
?
N-acetyl-Phe-tRNA + H2O
N-acetyl-Phe + tRNA
show the reaction diagram
N-acetyl-Phe-Val-tRNA + H2O
N-acetyl-Phe-Val + tRNA
show the reaction diagram
-
-
-
-
?
N-acetyl-Ser-tRNA + H2O
N-acetyl-Ser + tRNA
show the reaction diagram
N-acetyl-Trp-tRNA + H2O
N-acetyl-Trp + tRNA
show the reaction diagram
-
-
-
-
?
N-acetyl-Tyr-tRNA + H2O
N-acetyl-Tyr + tRNA
show the reaction diagram
-
-
-
-
?
N-acetyl-Val-tRNA + H2O
N-acetyl-Val + tRNA
show the reaction diagram
N-benzoyl-Gly-Gly-Phe-tRNA + H2O
N-benzoyl-Gly-Gly-Phe + tRNA
show the reaction diagram
-
-
-
-
?
N-benzoyl-Gly-GlyGly-Phe-tRNA + H2O
N-benzoyl-Gly-Gly-Gly-Phe + tRNA
show the reaction diagram
-
-
-
-
?
N-carbobenzyloxy-Phe-tRNA + H2O
N-carbobenzyloxy-Phe + tRNA
show the reaction diagram
-
-
-
-
?
N-formyl-Val-tRNA + H2O
N-formyl-Val + tRNA
show the reaction diagram
N-substituted aminoacyl-tRNA + H2O
N-substituted amino acid + tRNA
show the reaction diagram
oligolysyl-tRNA + H2O
oligo-Lys + tRNA
show the reaction diagram
-
-
-
-
?
Oregon Green-methionine-tRNA + H2O
Oregon Green-methionine + tRNA
show the reaction diagram
-
-
-
?
peptidyl-tRNA + H2O
?
show the reaction diagram
peptidyl-tRNA + H2O
peptide + tRNA
show the reaction diagram
peptidyl-tRNA hydrolase cleaves the ester bond between tRNA and the attached peptide in peptidyl-tRNA in order to avoid the toxicity resulting from its accumulation and to free the tRNA available for further rounds in protein synthesis
-
-
?
peptidyl-tRNAL + H2O
peptide + tRNA
show the reaction diagram
peptidyl-tRNALys + H2O
peptide + tRNALys
show the reaction diagram
Phe-Phe-tRNA + H2O
Phe-Phe + tRNA
show the reaction diagram
-
-
-
-
?
Phe-tRNA + H2O
Phe + tRNA
show the reaction diagram
phenyllactyl-Phe-tRNA + H2O
phenyllactyl-Phe + tRNA
show the reaction diagram
-
-
-
-
?
poly-Val-tRNA + H2O
poly-Val + tRNA
show the reaction diagram
-
-
-
-
?
Ser-tRNA + H2O
Ser + tRNA
show the reaction diagram
Ser-tRNAAla + H2O
Ser + tRNAAla
show the reaction diagram
-
-
-
?
Tyr-tRNA + H2O
Tyr + tRNA
show the reaction diagram
-
-
-
-
?
Val-tRNA + H2O
Val + tRNA
show the reaction diagram
Val-tRNAVal + H2O
Val + tRNAVal
show the reaction diagram
-
-
-
-
?
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
diacetyl-lysyl-tRNALys + H2O
diacetyl-lysine + tRNALys
show the reaction diagram
N-substituted aminoacyl-tRNA + H2O
N-substituted amino acid + tRNA
show the reaction diagram
peptidyl-tRNA + H2O
peptide + tRNA
show the reaction diagram
P9WHN7
peptidyl-tRNA hydrolase cleaves the ester bond between tRNA and the attached peptide in peptidyl-tRNA in order to avoid the toxicity resulting from its accumulation and to free the tRNA available for further rounds in protein synthesis
-
-
?
peptidyl-tRNAL + H2O
peptide + tRNA
show the reaction diagram
-
Pth is a key protein at the crossroads to the function of several translational factors, accumulation of peptidyl-tRNA in the cells leads to depletion of aminoacyl-tRNA pools and halts protein biosynthesis, it is vital for cells to maintain Pth activity to deal with the pollution of peptidyl-tRNAs generated during the initiation, elongation and termination steps of protein biosynthesis, overview
-
-
?
peptidyl-tRNALys + H2O
peptide + tRNALys
show the reaction diagram
-
accumulation of peptidyl-tRNA due to enzyme misfunction is toxic to the cells, overproduction of tRNALys suppresses the effects of pthTs mutation at 41C but not at 43C, and increases the levels of aminoacyl-tRNA
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-
?
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
-
activation is 25% lower than with Mg2+ or Mn2+
K+
-
0.3 mM, 77fold activation
KCl
-
300 mM, 77fold activation; activates about 80fold, optimally at 300 mM
MgCl2
-
40 mM, approx. 88fold activation
Zn2+
a zinc ion is coordinated by the conserved zinc-binding cluster in the C-domain, which is expected to be the enzymatic active site
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3'-deoxy-N[(O-methyl-L-tyrosyl)amino]adenosine
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-
3'-L-(N,N-diacetyl-lysinyl)amino-3'-deoxyadenosine
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-
70 S ribosome
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free N-carbobenzyloxy-Phe-tRNA is rapidly cleaved by the enzyme. When bound to a 30 S ribosome in the presence of poly(U), the substrate is hydrolyzed rapidly as when free. The addition of 50 S ribosomal subunits to form the 70S ribosomal binding complex protects the bound substrate from the enzyme
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deacetylated tRNA
diethyldicarbonate
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0.5 mM, 90% inactivation after 10 min, activity can be recovered to 41% of initial activity by treatment wit 200 mM hydroxylamine
non-esterified tRNALys
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-
-
tRNA-formyl-methionine
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from Escherichia coli
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tRNA-lysine
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from Escherichia coli
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tRNAfMet
-
; tRNAfMet from Escherichia coli
tRNALys
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tRNALys from Escherichia coli
Uncharged tRNA
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e.g. from Escherichia coli, at concentrations of 0.01 mM or above
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
spermidine
-
0.1 mM spermidine HCl, 73fold activation
spermidine-HCl
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0.1 mM, 73fold activation; activates about 80fold, optimally at 0.1 mM
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000016
acetyl-His-tRNAHis
0.008
bulk peptidyl-tRNA
-
at pH 5.0 and 25C
-
0.000003
dephosphorylated diacyl-lysine-tRNA
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50C, pH 7.5
-
0.00003
dephosphorylated formyl-methioninyl-tRNA
-
50C, pH 7.5
-
0.0000028 - 0.024
diacetyl-Lys-tRNALys
0.000011 - 0.006
diacetyl-lysine-tRNA
0.0000028 - 12.8
diacetyl-lysyl-tRNALys
0.000012
formyl-Met-tRNAfMet
-
pH 7.5, 50C
0.00003
formyl-Met-tRNAfMet, dephosphorylated
-
pH 7.5, 50C, dephosphorylated formyl-Met-tRNAfMet
-
0.000012
formyl-methionyl-tRNA
0.00003
formyl-methionyl-tRNAfMet
-
dephosphorylated substrate, pH 7.5, 50C, recombinant enzyme
0.00471 - 0.0269
N-acetyl-Ala-tRNA(Ala)
0.0022
N-acetyl-Met-tRNA
-
pH 7.0, 37C
0.0008
N-acetyl-Phe-tRNA
-
pH 7.5, 30C
0.000001
N-acetyl-phenylalanyl-tRNA
-
pH 7.0, 30C
0.0055
Oregon Green-methionine-tRNA
-
25C, pH 7.5
-
0.0008
Phe-tRNA
-
pH 7.5, 30C
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3.4
acetyl-His-tRNA
0.86 - 6.08
dephosphorylated diacyl-lysine-tRNA
3
dephosphorylated formyl-methioninyl-tRNA
Sulfolobus solfataricus
-
50C, pH 7.5
-
1.3
diacetyl-Lys-tRNA
Homo sapiens
Q9Y3E5
pH 7.5, 27C
0.028 - 3.6
diacetyl-Lys-tRNALys
1.3 - 3.6
diacetyl-lysine-tRNA
0.002 - 2.2
diacetyl-lysyl-tRNALys
3
formyl-Met-tRNAfMet
5.8 - 11.7
N-acetyl-Ala-tRNA(Ala)
0.7
N-acetyl-Met-tRNA
Escherichia coli
-
pH 7.0, 37C
3
o-hydroxycinnamic acid
Sulfolobus solfataricus
-
50C, pH 7.5
9.3
Oregon Green-methionine-tRNA
Escherichia coli
-
25C, pH 7.5
-
additional information
additional information
Escherichia coli
-
effect of mutations altering the 1-72 pair of E. coli tRNAMetf on the turnover-number
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.51
2'(3')-O-L-(N,N-diacetyl-lysinyl)adenosine
Escherichia coli
-
wild type enzyme, 28C, 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 0.1 mM EDTA, 0.1 mM dithiothreitol
166174
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
12
3'-L-(N,N-diacetyl-lysinyl)amino-3'-deoxyadenosine
-
at pH 6.0, with 50 mM sodium acetate and 200 mM NaCl, at 28C
0.00005
non-esterified tRNALys
-
pH 7.5, 50C, recombinant enzyme
-
0.000095
tRNA
-
pH 7.5, 50C, unfractionated Escherichia coli tRNA; unfractionated tRNA from Escherichia coli, 50C, pH 7.5
0.0001
tRNA-formyl-methionine
-
tRNA-ormyl-methionine from Escherichia coli, 50C, pH 7.5
-
0.00005
tRNA-lysine
-
tRNA-lysine from Escherichia coli, 50C, pH 7.5
-
0.00011
tRNAfMet
0.00005
tRNALys
-
pH 7.5, 50C, tRNALys from Escherichia coli
0.0001
Uncharged tRNA
-
pH 7.5, 50C, recombinant enzyme
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0149
-
-
0.078
-
substrate D-tyrosyl-tRNA, 37C, pH 7.5
0.15
-
substrae D-tyrosyl-tRNA
4.14
-
; purified recombinant enzyme
30
-
substrate diacetyl-lysine-tRNA
31.8
-
substrate diacetyl-lysine tRNA, 37C, pH 7.5
additional information
-
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 7.5
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-
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 8.5
approx. 38% at pH 6.0, approx. 20% of maximal activity at pH 8.5
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10
-
isoelectric focusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
enzyme from encysted embryos is specific for acetyl-Phe-tRNA. An unspecific hydrolase active on several N-substituted amiinoacyl-tRNAs is practically absent in the encysted embryos and during embryogenesis and appears abruptly during larval development
Manually annotated by BRENDA team
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paraproerythroblast, low activity
Manually annotated by BRENDA team
-
-
Manually annotated by BRENDA team
-
-
Manually annotated by BRENDA team
-
low activity
Manually annotated by BRENDA team
-
low activity
Manually annotated by BRENDA team
additional information
-
mutant strain AA7852 with temperature-sensitive Pth grown at 32C and at 41C
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Archaeoglobus fulgidus (strain ATCC 49558 / VC-16 / DSM 4304 / JCM 9628 / NBRC 100126)
Archaeoglobus fulgidus (strain ATCC 49558 / VC-16 / DSM 4304 / JCM 9628 / NBRC 100126)
Burkholderia thailandensis (strain E264 / ATCC 700388 / DSM 13276 / CIP 106301)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Francisella tularensis subsp. tularensis (strain SCHU S4 / Schu 4)
Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440)
Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155)
Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155)
Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155)
Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Salmonella typhimurium (strain 14028s / SGSC 2262)
Streptococcus pyogenes serotype M49 (strain NZ131)
Sulfolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
Vibrio cholerae serotype O1 (strain M66-2)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20460
calculated from amino acid sequence
23000
SDS-PAGE
25300
dynamic light-scattering
46000
-
gel filtration
55000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
native enzyme and in complex with cytidine or uridine, hanging drop vapor diffusion method, using 25% (w/v) PEG 10000, 0.3 M MgCl2 and 0.1 M HEPES buffer at pH 6.0
crystal structure analysis
-
crystal structure at 1.3 A resolution
-
crystallization by using polyethylene glycol as precipitant, recombinant enzyme
-
crystallization using polyethylene glycol as precipitant and isopropanol as additive, crystal structure at 1.2 A
in complex with the tRNA CCA-acceptor-TpsiC domain of tRNAAla, sitting drop vapor diffusion method, using 100 mM sodium acetate buffer (pH 5.2), 20% (w/v) 1,4-butanediol and 30 mM glycyl-glycylglycine, at 20C
-
recombinant enzyme, sitting drop vapor diffusion techniques; sitting-drop vapor diffusion at room temperature, 10 mg/ml protein in 100 mM HEPES, pH 7.5 and 20% polyethylene glycol 10000 is equilibrated against a reservoir of the same solution, crystals diffract to 2.0 A
hanging drop vapor diffusion method, using 30% (w/v) PEG-1500 and 10% (v/v) isopropanol in 100 mM HEPES buffer, pH 6.5
-
microbatch-under-oil method, using 0.1 M HEPES pH 7.5, 15% (w/v) PEG 8000, 5% (v/v) isopropanol or 0.1 M HEPES pH 7.5 and 5% (v/v) dioxane with 25% (w/v) PEG 8000
purified recombinant enzyme, X-ray diffraction structure determination and analysis at 1.98 A, 2.35 A, and 2.49 A resolution, molecular replacement
purified recombinant His-tagged enzyme, microbatch method, 0.002 ml each of the protein solution, containing 6 mg/ml protein, 20 mM Tris-HCl, pH 7.5, 100 mM NaCl and 2 mM 2-mercaptoethanol, and the precipitant solution, containing 20% w/v PEG 8000 in 0.1 M HEPES, pH 7.5, and 5% v/v 2-propanol or dioxane or 25% w/v PEG 8000, 100 mM sodium cacodylate, pH 6.6, and 5% v/v 2-propanol, are mixed, 5 days to 2 weeks, X-ray diffraction structure determination and analysis at 1.97-2.49 A resolution
-
native enzyme and in complex with 3'-deoxy-N[(O-methyl-L-tyrosyl)amino]adenosine or 5-azacytidine, hanging drop vapor diffusion method, using 25% (w/v) PEG 4000, 5% (v/v) propan-2-ol and 0.1 M HEPES buffer, pH 7.5
hanging-drop vapour-diffusion method at 20C, crystal structure is determined at 2.7 A resolution
hanging drop vapor diffusion method, using 0.03 M citric acid, 0.05 M Bis-Tris propane, 1% (v/v) glycerol, 3% (w/v) sucrose, 25% (w/v) PEG 6000 pH 7.6
D0ZJ57
hanging drop vapour diffusion method, 1.8 A resolution
-
purified recombinant enzyme, hanging drop vapour diffusion method, 24C, 0.0027 ml of 1.3 mg/ml protein in 20 mM Tris-HCl, pH 7.0, 0.1 mM EDTA, and 10 mM 2-mercaptoethanol are mixed with 0.0007 ml of 11 mg/ml tRNAfMet solution and 0.002 ml of reservoir solution containing 0.8 M LiSO4 and 1.6% PEG 8000, crystallization of tRNA free crystals within a few days, X-ray diffraction structure determination and analysis of native and HgBr2-containing crystals at 1.8-3.0 A resolution, modeling
sitting drop vapor diffusion method, using 100 mM phosphate-citrate buffer pH 4.2, and 50% (w/v) 2-methyl-2,4-pentanediol
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 7.5
-
4C, 24 h, stable
648113
6 - 8
-
30C, 30 min, stable
648114
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
-
10 min, 70% loss of activity
50
-
10 min, 85% loss of activity
additional information
-
thermally-induced unfolding curve for MtPth indicates a simple two-state unfolding process without any intermediates, thermodynamic stability of the enzyme, pH 6.5, 25C, overview
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
urea/guanidinium chloride-induced unfolding curve for MtPth indicates a simple two-state unfolding process without any intermediates, pH 6.5, 25C
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-10C, 50% glycerol, purified enzyme is stable for several months
-
-10C, in presence of 1 mg/ml bovine serum albumin, no loss of activity
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-20C, buffer solution: 0.05 M Tris-HCl, pH 7.0, 0.01 M 2-mercaptoethanol, 0.01 M magnesium acetate, or as an ammonium sulfate precipitate, 2 months without significant loss of activity
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22C, 20 mM Bis-Tris pH 6.6, 50 mM NaCl, 2 mM dithiothreitol, several weeks, without loss of activity or precipitation
D0ZJ57
22C, 20 mM sodium acetate, 150 mM sodium chloride, 2 mM dithiothreitol, pH 5.0, several months, minimal loss of activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; recombinant PTH, heat, ammonium sulfate, Superdex 75, HI-Propyl, SP-Sepharose; recomninant PTH from Escherichia coli strain XL 1-Blue 8500fold by ultracentrifugation, heat treatment, ammonium sulfate fractionation, gel filtration, hydrophobic interaction chromatography, and ion exchange chromatography to homogeneity
-
ammonium sulfate precipitation
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HisTrap column chromatography, Ni-NTA agarose column chromatography, and Q Sepharose column chromatography
-
HisTrap nickel affinity column chromatography and Superdex 75 gel filtration
-
Ni-chelating column chromatography
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Ni-NTA affinity resin column chromatography and Superdex G50 gel filtration
Ni-NTA column chromatography and Superdex G-75 gel filtration
Ni2+-chelating affinity column chromatography and Superose 12 gel filtration
D0ZJ57
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity and anion exchange chromatography
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recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and dialysis
-
recombinant His-tagged Pth
recombinant Pth
-
Super Q Toyopearl 650M column chromatography, Resource Q column chromatography, and Superdex 75 gel filtration
wild-type and mutant enzymes
-
wild-type and mutant enzymes N10A, H20A, M67A, F66A, D93A, H113A, K142A
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed as GST-fusion protein in Escherichia coli Rosetta pLysS (GST later cleaved), or in human HEK293T cells as FLAG-tagged protein
expressed in Escherichia coli BL21 (DE3) pMGK cells with eight nonnative residues at the C-terminus (LEHHHHHH) to facilitate protein purification
expressed in Escherichia coli BL21 pLysS cells
D0ZJ57
expressed in Escherichia coli BL21(DE3) cells
expressed in Escherichia coli BL21(DE3)pLysS cells
-
expressed in Escherichia coli BL21(lambdaDE3) cells
expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL cells
expressed in Escherichia coli strain K37DELTArecAlambdaDE3
-
expression in Escherichia coli
expression in Escherichia coli; full length human Pth2 clone as tempolate , subcloned into the NcoI/BamHI sites of the pET15b bacterial expression vector. The ligated plasmid is transfoirmed into Escherichia coli BL21(DE3) strain
expression in Escherichia coli; gene SS00175, DNA and amino acid sequence determination and analysis, complementation of an enzyme-deficient Escherichia coli mutant strain, and of two Saccharomyces cerevisiae gene YHR189w or YBL057c disruption mutants, overview, expression of PTH in Escherichia coli strain XL 1-Blue
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gene pth, orf Rv1014c, DNA and amino acid sequence determination and analysis, expression of wild-type and mutant C-terminally His-tagged enzymes in Escherichia coli strain BL21(DE3) and in Escherichia coli thermosensitive strain AA7852, the mutant strain is able to grow at the nonpermissive temperature of 42C, at 39C, overexpression of MtPth in AA7852 cells allowed the cells to remain viable in the presence of up to 200 mg/ml erythromycin, overview
-
gene pth, orf Rv1014c, DNA and amino acid sequence determination, expression of the His-tagged enzyme in Escherichia coli strain BL21(DE3)
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pth2 gene, expression in Escherichia coli
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D93A
turnover-number is 0.1% of the turnover-number for wild-type enzyme, Km-value for diacetyl-Lys-tRNALys is 1.67fold higher than the Km-value for the wild-type enzyme
D93N
-
4% of wild-type kcat
F66A
turnover-number is 26% of the turnover-number for wild-type enzyme, Km-value for diacetyl-Lys-tRNALys is 1.15fold higher than the Km-value for the wild-type enzyme
H113A
turnover-number is 33% of the turnover-number for wild-type enzyme, Km-value for diacetyl-Lys-tRNALys is 1.46fold higher than the Km-value for the wild-type enzyme
H188A
-
the mutation results in a 5.4fold decrease in the kcat/Km value compared to the wild type enzyme
H20N
-
no activity
H20Q
-
no activity
K103Q
-
54% of wild-type kcat
K103R
-
68% of wild-type kcat
K103S
-
28% of wild-type kcat
K105Q
-
20% of wild-type kcat
K105R
-
26% of wild-type kcat
K105S
-
16% of wild-type kcat
K113Q
-
98% of wild-type kacat
K142A
turnover-number is 24% of the turnover-number for wild-type enzyme, Km-value for diacetyl-Lys-tRNALys is 4fold higher than the Km-value for the wild-type enzyme
M67A
turnover-number is 4.7% of the turnover-number for wild-type enzyme, Km-value for diacetyl-Lys-tRNALys is 70% of the Km-value for the wild-type enzyme
M67E
-
0.5% of wild-type kcat
N185A
-
the mutation results in a 5.7fold decrease in the kcat/Km value compared to the wild type enzyme
N185A/H188A
-
the mutation results in a 7.7fold decrease in the kcat/Km value compared to the wild type enzyme
C166A
-
site-directed mutagenesis, the mutant effectively complements the enzyme-defective thermosensitive Escherichia coli mutant strain AA7852 for growth at 42C
C67S
-
site-directed mutagenesis, the mutant effectively complements the enzyme-defective thermosensitive Escherichia coli mutant strain AA7852 for growth at 42C
D95N
-
site-directed mutagenesis, the catalytic residue mutant is not able to complement the enzyme-defective thermosensitive Escherichia coli mutant strain AA7852 for growth at 42C
H22N
-
site-directed mutagenesis, the catalytic residue mutant is not able to complement the enzyme-defective thermosensitive Escherichia coli mutant strain AA7852 for growth at 42C; site-directed mutagenesis, the mutation affects the enzyme structure, overview
N12D
-
site-directed mutagenesis, the catalytic residue mutant is not able to complement the enzyme-defective thermosensitive Escherichia coli mutant strain AA7852 for growth at 42C
C166A
-
site-directed mutagenesis, the mutant effectively complements the enzyme-defective thermosensitive Escherichia coli mutant strain AA7852 for growth at 42C
-
C67S
-
site-directed mutagenesis, the mutant effectively complements the enzyme-defective thermosensitive Escherichia coli mutant strain AA7852 for growth at 42C
-
D95N
-
site-directed mutagenesis, the catalytic residue mutant is not able to complement the enzyme-defective thermosensitive Escherichia coli mutant strain AA7852 for growth at 42C
-
H22N
-
site-directed mutagenesis, the catalytic residue mutant is not able to complement the enzyme-defective thermosensitive Escherichia coli mutant strain AA7852 for growth at 42C; site-directed mutagenesis, the mutation affects the enzyme structure, overview
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C703A
mutation abolishes editing activity
D86A
-
kcat/Km for diacetyl-lysyl-tRNALys is 0.22% of wild-type value
D86A/K18A
-
kcat/Km for diacetyl-lysyl-tRNALys is 0.17% of wild-type value
H25A
-
kcat/Km for diacetyl-lysyl-tRNALys is 13% of wild-type value
K118A
-
kcat/Km for diacetyl-lysyl-tRNALys is 87% of wild-type value
K18A
-
kcat/Km for diacetyl-lysyl-tRNALys is 0.2% of wild-type value
K56A
-
kcat/Km for diacetyl-lysyl-tRNALys is 12% of wild-type value
Q22A
-
kcat/Km for diacetyl-lysyl-tRNALys is 20% of wild-type value
Q54A
-
kcat/Km for diacetyl-lysyl-tRNALys is 47% of wild-type value
T90A
-
kcat/Km for diacetyl-lysyl-tRNALys is 1.4% of wild-type value
T98A
-
kcat/Km for diacetyl-lysyl-tRNALys is 34% of wild-type value
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
-
Pth is a potential drug target to control eubacterial infections
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