Information on EC 2.1.1.74 - methylenetetrahydrofolate-tRNA-(uracil54-C5)-methyltransferase (FADH2-oxidizing)

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.1.1.74
-
RECOMMENDED NAME
GeneOntology No.
methylenetetrahydrofolate-tRNA-(uracil54-C5)-methyltransferase (FADH2-oxidizing)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
5,10-methylenetetrahydrofolate + uracil54 in tRNA + FADH2 = tetrahydrofolate + 5-methyluracil54 in tRNA + FAD
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
methyl group transfer
-
-
-
-
reductive methylation
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
5,10-methylenetetrahydrofolate:tRNA (uracil54-C5)-methyltransferase
Up to 25% of the bases in mature tRNA are post-translationally modified or hypermodified. One almost universal post-translational modification is the conversion of U54 into ribothymidine in the TPsiC loop, and this modification is found in most species studied to date [2]. Unlike this enzyme, which uses 5,10-methylenetetrahydrofolate and FADH2 to supply the atoms for methylation of U54, EC 2.1.1.35, tRNA (uracil54-C5)-methyltransferase, uses S-adenosyl-L-methionine.
CAS REGISTRY NUMBER
COMMENTARY hide
56831-74-4
74665-78-4 deleted
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Clostridium acidi-urici
-
-
-
Manually annotated by BRENDA team
no activity in Micrococcus luteus
strains ATCC 4698 and ATCC12698, formerly Micrococcus lysodeikticus
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Treponema palladium
-
-
-
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
5,10-methylenetetrahydrofolate + tRNA containing uridine at position 54 + FADH2
tetrahydrofolate + tRNA containing ribothymidine at position 54 + FAD
show the reaction diagram
5,10-methylenetetrahydrofolate + tRNA UpsiC + FADH2
tetrahydrofolate + tRNA TpsiC + FAD
show the reaction diagram
5,10-methylenetetrahydrofolate + tRNA UpsiC + FADH2
tetrahydrofolate + tRNA TpsiC + FAD+
show the reaction diagram
5,10-methylenetetrahydrofolate + tRNA UpsiC + NADH
tetrahydrofolate + tRNA TpsiC + NAD+
show the reaction diagram
-
-
-
-
?
N5,N10-methylenetetrahydrofolate + tRNA containing uridine at position 54 + FADH2
tetrahydrofolate + tRNA containing ribothymidine at position 54 + FAD
show the reaction diagram
-
-
-
-
?
S-adenosyl-L-methionine + tRNAPhe containing uridine at position 54
S-adenosyl-L-homocysteine + tRNAPhe containing ribothymidine at position 54
show the reaction diagram
S-adenosyl-L-methionine + tRNAPhe mutant A58G containing uridine at position 54
S-adenosyl-L-homocysteine + tRNAPhe mutant A58G containing ribothymidine at position 54
show the reaction diagram
S-adenosyl-L-methionine + tRNAPhe mutant A58U containing uridine at position 54
S-adenosyl-L-homocysteine + tRNAPhe mutant A58U containing ribothymidine at position 54
show the reaction diagram
S-adenosyl-L-methionine + tRNAPhe mutant G57A containing uridine at position 54
S-adenosyl-L-homocysteine + tRNAPhe mutant G57A containing ribothymidine at position 54
show the reaction diagram
S-adenosyl-L-methionine + tRNAPhe nucleotides 18-76 containing uridine at position 54
S-adenosyl-L-homocysteine + tRNAPhe nucleotides 18-76 containing ribothymidine at position 54
show the reaction diagram
-
-
-
?
S-adenosyl-L-methionine + tRNAPhe nucleotides 44-76 containing uridine at position 54
S-adenosyl-L-homocysteine + tRNAPhe nucleotides 44-76 containing ribothymidine at position 54
show the reaction diagram
-
-
-
?
S-adenosyl-L-methionine + tRNAPhe with deletions of aminoacyl stem and D-arm containing uridine at position 54
S-adenosyl-L-homocysteine + tRNAPhe with deletions of aminoacyl stem and D-arm containing ribothymidine at position 54
show the reaction diagram
-
-
-
?
S-adenosyl-L-methionine + tRNAPhe with deletions of aminoacyl stem containing uridine at position 54
S-adenosyl-L-homocysteine + tRNAPhe with deletions of aminoacyl stem containing ribothymidine at position 54
show the reaction diagram
-
-
-
?
S-adenosyl-L-methionine + tRNAPhe with deletions of anticodon arm containing uridine at position 54
S-adenosyl-L-homocysteine + tRNAPhe with deletions of anticodon armand D-arm containing ribothymidine at position 54
show the reaction diagram
-
-
-
?
S-adenosyl-L-methionine + tRNAPhe with deletions of D-arm containing uridine at position 54
S-adenosyl-L-homocysteine + tRNAPhe with deletions of D-arm containing ribothymidine at position 54
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
5,10-methylenetetrahydrofolate + tRNA containing uridine at position 54 + FADH2
tetrahydrofolate + tRNA containing ribothymidine at position 54 + FAD
show the reaction diagram
5,10-methylenetetrahydrofolate + tRNA UpsiC + FADH2
tetrahydrofolate + tRNA TpsiC + FAD
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADH
-
is used in the assay as electron donor
NADPH
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.5
5,10-methylenetetrahydrofolate
-
-
0.0011
tRNAPhe containing uridine at position 54
pH not specified in the publication, 60C
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0.0022
tRNAPhe mutant A58G containing uridine at position 54
pH not specified in the publication, 60C
-
0.0013
tRNAPhe mutant A58U containing uridine at position 54
pH not specified in the publication, 60C
-
0.0021
tRNAPhe mutant G57A containing uridine at position 54
pH not specified in the publication, 60C
-
0.0045
tRNAPhe nucleotides 18-76 containing uridine at position 54
pH not specified in the publication, 60C
-
0.0026
tRNAPhe nucleotides 44-76 containing uridine at position 54
pH not specified in the publication, 60C
-
0.0019
tRNAPhe with deletions of aminoacyl stem and D-arm containing uridine at position 54
pH not specified in the publication, 60C
-
0.0013
tRNAPhe with deletions of aminoacyl stem containing uridine at position 54
pH not specified in the publication, 60C
-
0.0012
tRNAPhe with deletions of anticodon arm containing uridine at position 54
pH not specified in the publication, 60C
-
0.0015
tRNAPhe with deletions of D-arm containing uridine at position 54
pH not specified in the publication, 60C
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8
-
assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
assay at
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
33600
-
untagged protein
47900
-
His-tagged fusion protein
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
free TrmFO or bound to tetrahydrofolate-bound or glutathione, addition of GSH greatly improves the quality of the crystals, X-ray diffraction structure determinationand analysis at 2.1 A, 1.6 A, and 1.05 A resolutions, respectively
purified recombinant enzyme, from 25% v/v PEG 4000, 100 mM NaCl and sodium citrate buffer, pH 5.0, at 291 K using the hanging-drop vapor-diffusion method in 0.0016 ml with 8 mg/ml protein, 7 days, X-ray diffraction structure determination and analysis at 2.6 A resolution
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
enzyme requires FAD+ for stability
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified to homogeneity by metal affinity or ion exchange and heparin affinity, respectively, followed by gel filtration
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recombinant enzyme from Escherichia coli strain BL21(DE3) by anion and cation exchange chromatography and gel filtration to over 95% purity
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recombinant enzyme from Escherichia coli to homogeneity
using Ni-NTA chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli as a His-tagged fusion protein
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expression in Escherichia coli
overexpression in Escherichia coli strain BL21(DE3)
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the encoded recombinant protein contains tightly bound flavin and is active in Escherichia coli mutant lacking m5U-54 in tRNAs and in vitro using T7 tRNA transcript as substrate
untagged N-terminus and C-terminus (His)6-tagged TrmFO from Bacillus subtilis is expressed in Escherichia coli. The tag does not significantly alter the expression level, flavin content, activity and secondary structure of the protein
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C193A
-
mutant is nearly as active as the wild-type enzyme
C226A
-
mutant loses both the tRNA methylation activity and the capacity to form a covalent complex with the 5-FU-mini-RNA
C53A
-
mutant is inactive but like the wild-type enzyme, mutant C53A is capable of forming a covalent complex with a 5-fluorouridine-containing mini-RNA. Mutation of Cys-53 changes the accessibility of the FAD-binding site and impairs the conformational stability of TrmFO
C53A/C226A
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as for the single C226A mutant, no protein-RNA covalent complex is detectable with the double mutant
R312G
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mutant isolated after cloning is found to be defective in tRNA methylation, with an activity corresponding only to 40% that of the wild-type enzyme
S54A
-
mutant is nearly as active as the wild-type enzyme
C223A
site-directed mutagenesis, almost inactive mutant
C51A
site-directed mutagenesis, almost inactive mutant
E341A
site-directed mutagenesis, the active mutant shows a decrease in both FAD binding and methylation activity compared to the wild-type enzyme
H308A
site-directed mutagenesis, the mutant shows 57% of wild-type enzyme activity compared to the wild-type enzyme
K282A
site-directed mutagenesis, almost inactive mutant
K287A
site-directed mutagenesis, almost inactive mutant
K409A
site-directed mutagenesis, the mutant shows decreased activity compared to the wild-type enzyme
K410A
site-directed mutagenesis, the mutant shows decreased activity compared to the wild-type enzyme
N310A
site-directed mutagenesis, the mutant shows 23% of wild-type enzyme activity compared to the wild-type enzyme
R97A
site-directed mutagenesis, the mutant shows decreased activity compared to the wild-type enzyme
W283A
site-directed mutagenesis, the mutant shows slightly decreased activity
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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