Information on EC 2.1.1.35 - tRNA (uracil54-C5)-methyltransferase

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

EC NUMBER
COMMENTARY hide
2.1.1.35
-
RECOMMENDED NAME
GeneOntology No.
tRNA (uracil54-C5)-methyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
S-adenosyl-L-methionine + uracil54 in tRNA = S-adenosyl-L-homocysteine + 5-methyluracil54 in tRNA
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
methyl group transfer
SYSTEMATIC NAME
IUBMB Comments
S-adenosyl-L-methionine:tRNA (uracil54-C5)-methyltransferase
Unlike this enzyme, EC 2.1.1.74 (methylenetetrahydrofolate---tRNA-(uracil54-C5)-methyltransferase (FADH2-oxidizing)), uses 5,10-methylenetetrahydrofolate and FADH2 to supply the atoms for methylation of U54 [4].
CAS REGISTRY NUMBER
COMMENTARY hide
37257-02-6
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
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Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain 1100/K12
-
-
Manually annotated by BRENDA team
strain C600/pTN106
-
-
Manually annotated by BRENDA team
strain GB-1-5-39/pTN102
-
-
Manually annotated by BRENDA team
strain GRB822
-
-
Manually annotated by BRENDA team
strain HB101/pMTXR1
-
-
Manually annotated by BRENDA team
strain K12MO7
-
-
Manually annotated by BRENDA team
strain SN01
-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
S-adenosyl-L-methionine + 16S rRNA
S-adenosyl-L-homocysteine + 16S rRNA containing thymine
show the reaction diagram
S-adenosyl-L-methionine + DNA analog of the T-stem/loop domain of yeast tRNAPhe
S-adenosyl-L-homocysteine + DNA analog of the T-stem/loop domain of yeast tRNAPhe containing thymine
show the reaction diagram
-
DNA analog of the T-stem/loop domain of yeast tRNAPhe composed of d(A,U,G,C) is also a substrate, slow methylation of tDNAPheT
-
?
S-adenosyl-L-methionine + tRNA containing uridine at position 54
S-adenosyl-L-homocysteine + tRNA containing ribothymidine at position 54
show the reaction diagram
S-adenosyl-L-methionine + uridine54 in tRNA
S-adenosyl-L-homocysteine + 5-methyluridine54 in tRNA
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
S-adenosyl-L-methionine + tRNA containing uridine at position 54
S-adenosyl-L-homocysteine + tRNA containing ribothymidine at position 54
show the reaction diagram
S-adenosyl-L-methionine + uridine54 in tRNA
S-adenosyl-L-homocysteine + 5-methyluridine54 in tRNA
show the reaction diagram
additional information
?
-
Q9UZR7
the enzyme emerged through an ancient horizontal transfer of an RNA (uracil, C5)-methyltransferase-like gene from bacteria to archaea
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-
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
S-adenosyl-L-methionine
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ammonium acetate
-
30% activation at 50 mM
Ca2+
-
activates
Fe2+
-
the enzyme contains an [Fe4S4] cluster, overview
Mn2+
-
activates
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5-Fluorouracil substituted tRNA
-
DNA analog of the T-stem/loop domain
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DNA analog of the T-stem/loop domain of yeast tRNAPhe composed of d(A,U,G,C) inhibits the methylation of unfractionated methyl-deficient E. coli tRNA, significant inhibition when the concentration of tDNAPheT is twice that of the tRNA
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ethylthioadenosine
-
0.2 mM
K+
-
inhibitory in presence of Mg2+
L-ethionine
-
selective inhibitor of uracil tRNA methylase, non-competitive to S-adenosyl-L-methionine
L-methionine
-
30 mM, weak
mature tRNA
-
weak, competitive inhibitor to methyl-deficient tRNA, non-competitive to S-adenosyl-L-methionine
Mg2+
-
inhibits at low concentrations
Mn2+
-
inhibits at low concentrations
Na+
-
inhibitory in presence of Mg2+
NO2Ura-tRNA
-
potent mechanism-based inhibitor, competitive with respect to tRNA, can be reversed by dilution into a large excess of tRNA substrate, forms a reversible covalent complex in which Cys-324 of RUMT is linked to the 6-position of NO2-Ura 54 in NO2-Ura-tRNA, mechanism
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norleucine
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30 mM, weak
p-chloromercuribenzoate
p-hydroxymercuribenzoate
-
-
putrescine
-
inhibition at optimal Mg2+-concentration
ribonuclease
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inactivates
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S-adenosyl-L-ethionine
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0.6 mM
S-adenosyl-L-homocysteine
spermidine
-
inhibition at optimal Mg2+-concentration
Spleen phosphodiesterase
-
weak
-
structural analogues of S-adenosyl-L-homocysteine
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tRNAPhe
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inhibits binding of 16S rRNA to RUMT
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
dithiothreitol
-
activates
glutathione
putrescine
-
activates, inhibitory at optimal Mg2+ concentration
spermidine
spermine
-
slight activation
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0037 - 0.004
11mer of the T-arm of E. coli tRNA1Val
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0.005
17mer of the T-arm of E. coli tRNA1Val
-
-
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0.0145
DNA analog of the T-stem/loop domain of yeast tRNAPhe
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-
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0.025
methyl deficient tRNA
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-
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0.29
methyl deficient tRNA from E. coli
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-
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0.002 - 0.018
S-adenosyl-L-methionine
0.0028
unmodified E. coli tRNA1Val
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-
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0.0057
unmodified T-stem/loop domain of yeast tRNAPhe
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-
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0.0156
unmodified yeast tRNAPhe TPsiC-stem and loop heptadecamer
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-
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0.00008
uridine54 in tRNA
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0.0011
wheat germ tRNA1Gly
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-
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0.00037 - 0.0008
yeast tRNAPhe
-
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0015
11mer of the T-arm of E. coli tRNA1Val
Escherichia coli
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-
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0.0333
17mer of the T-arm of E. coli tRNA1Val
Escherichia coli
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-
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0.048
unmodified E. coli tRNA1Val
Escherichia coli
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-
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0.317
uridine54 in tRNA
Escherichia coli
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0.0317 - 0.0917
yeast tRNAPhe
-
additional information
additional information
Escherichia coli
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values for several mutants of yeast tRNAPhe
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.03 - 0.1
mature tRNA
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-
0.001
NO2Ura-tRNA
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-
-
0.0024 - 0.02
S-adenosyl-L-homocysteine
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.000167
-
-
0.00134
-
-
0.00165
-
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
-
assay at
7.5 - 8.5
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Tris-HCl buffer
8.5 - 9
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triethanolamine buffer
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.8 - 8.8
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25% inhibition of enzyme activity at pH 7.8 and 8.8
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35
-
assay at
38
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assay at
80
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kinetics of m5U formation by PAB0760 proceed faster at 80°C than at 50°C
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
the m5U modification is found in unspliced tRNAs, suggesting that enzyme action is within the nucleus
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Pyrococcus abyssi (strain GE5 / Orsay)
Pyrococcus abyssi (strain GE5 / Orsay)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
38000
-
gel filtration
62000
-
during the synthesis of m5U54 a covalent 62 kDa TrmA-tRNA intermediate is formed, determined by SDS-PAGE and immunoblotting
65000
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glycerol gradient centrifugation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
the enzyme contains an N-terminal TRAM domain, structure-function analysis, overview
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
the structure of Escherichia coli E358Q 5-methyluridine 54 tRNA methyltransferase, in a covalent complex with a 19 nt T arm analog, is solved to 2.4 A resolution
native and selenomethionylated purified enzyme PabTrmU54 in complex with S-adenosyl-L-homocysteine, hanging-drop by vapor diffusion method, 0.001 ml of a mixture of protein and S-adenosyl-L-methionine in a 1:2 molar ratiomixed with 0.001 ml of 0.6 ml reservoir solution containing 15% PEG 8000, 0.05 M ammonium sulfate, 0.1 M sodium citrate, pH 5.6,18°C, few days, X-ray diffraction structure determination and analysi at 1.9 A resolution, molecular replacement, modelling
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
-
2 h, stable
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
freeze-thawing inactivates
-
freezing without antifreeze agents inactivates, 30% ethylene glycol at -20°C stabilizes, dialysis inactivates, best pH used for dialysis is 6.5
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ethylene glycol
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70°C, at least 6 months, stable
-
-85°C, several months, stable
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0°C, partially purified, 48 h, about 30% loss of activity
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0°C, purified, 6 months, stable
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4°C, crude extract, 10 mM potassium phosphate, pH 7, 1 mM EDTA, 0.1 mM dithiothreitol, 10% v/v ethylene glycol, 0.02% sodium azide, 7 days, 10% loss of activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
3 enzyme forms
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5800fold purification
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affinity elution and hydroxylapatite chromatography
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may be 3 isoenzymes
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may be 3 isoenzymes; partial purification, hydroxylapatite chromatography
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native and selenomethionylated enzyme by nickel affinity chromatography
polyethyleneimine precipitation, phosphocellulose and affinity chromatography
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purification of glutathione S-transferase tagged recombinant, Trm2p, expressed in Escherichia coli BL21
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rapid purification scheme
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the His6 tag protein is purified using a Talon column, the tag is cleaved by thrombin, thrombin is removed by passage over a benzamidine column
using Ni-NTA chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning and sequencing of the TRM2 gene encoding enzyme, gene is previously identified as RNC1/NUD1 gene, but does not encode the yNucR endo-exonuclease, TRM2 gene is not essential for cell viability and contains alternative translational start sites, glutathione S-transferase tagged recombinant, Trm2p, is expressed in Escherichia coli
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enzyme is encoded by the nonessential TRM2 gene
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expressed in Escherichia coli as a His-tagged fusion protein
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genes PAB0719 and PAB0760, DNA and amino acid sequence determination and analysis, detailed phylogenetic analysis, expression in Escherichia coli strain BL21(DE3)
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hybrid plasmid carrying trmA gene encoding enzyme is used to amplify enzyme production, trmA gene is sequenced
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into the pET28b expression vector
the plasmids pGP100, pDM4, pKD4 and pKD46 are used for the construction of the mutants
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trmA gene encodes enzyme, gene is essential for the organism
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trmA gene encoding for RUMT is cloned into a pET vector and transformed into Escherichia coli BL21 (DE3) cells, extremely high-level expression system
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trmA gene is cloned under control of the inducible lac promoter, DH5alpha
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C324A
-
level of total TrmA-associated peptide 41%, amount of m5U in total tRNA 4%, relative to the levels found in the wild-type
D299A
-
level of total TrmA-associated peptide 63%, amount of m5U in total tRNA 1%, relative to the levels found in the wild-type
E358K
-
level of total TrmA-associated peptide 59%, amount of m5U in total tRNA 9%, relative to the levels found in the wild-type
E358Q
mutation of the catalytic base Glu-358 to Gln arrests catalysis and allows isolation of the covalent protein-RNA complex for crystallization
F188A
-
level of total TrmA-associated peptide 84%, amount of m5U in total tRNA 22%, relative to the levels found in the wild-type
G220D
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level of total TrmA-associated peptide 76%, amount of m5U in total tRNA 2%, relative to the levels found in the wild-type
G360D
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level of total TrmA-associated peptide 53%, amount of m5U in total tRNA 1%, relative to the levels found in the wild-type
Q190A
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level of total TrmA-associated peptide 77%, amount of m5U in total tRNA 14%, relative to the levels found in the wild-type
R302A
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level of total TrmA-associated peptide 82%, amount of m5U in total tRNA 87%, relative to the levels found in the wild-type
W132C
-
level of total TrmA-associated peptide 102%, amount of m5U in total tRNA 34%, relative to the levels found in the wild-type
W202C
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level of total TrmA-associated peptide 1%, amount of m5U in total tRNA 15%, relative to the levels found in the wild-type
C521A
-
mutant without methyltransferase activity, putative nucleophilic Cys-521 may be involved in Cys-U54 bond formation
G428D
-
mutant without methyltransferase activity, G-428 is located at the presumed S-adenosyl-L-methionine binding site
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
molecular biology
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