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Information on EC 2.1.1.74 - methylenetetrahydrofolate-tRNA-(uracil54-C5)-methyltransferase [NAD(P)H-oxidizing] and Organism(s) Bacillus subtilis and UniProt Accession P39815

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IUBMB Comments
A flavoprotein (FAD). 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 . Unlike this enzyme, which uses 5,10-methylenetetrahydrofolate and NAD(P)H to supply the atoms for methylation of U54, EC 2.1.1.35, tRNA (uracil54-C5)-methyltransferase, uses S-adenosyl-L-methionine.
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Bacillus subtilis
UNIPROT: P39815
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The taxonomic range for the selected organisms is: Bacillus subtilis
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Synonyms
trmfo, trna (m5u54)-methyltransferase, fdrts, folate-dependent ribothymidyl synthase, folate-dependent trna methyltransferase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tRNA:m5U-54 MTase
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folate-dependent ribothymidyl synthase
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-
-
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folate-dependent tRNA methyltransferase
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methylenetetrahydrofolate-transfer ribonucleate uracil 5-methyltransferase,
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-
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tRNA (m5U54)-methyltransferase
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-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
methyl group transfer
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-
-
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reductive methylation
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-
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SYSTEMATIC NAME
IUBMB Comments
5,10-methylenetetrahydrofolate:tRNA (uracil54-C5)-methyltransferase
A flavoprotein (FAD). 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 NAD(P)H 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
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 UpsiC + FADH2
tetrahydrofolate + tRNA TpsiC + FAD
show the reaction diagram
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-
-
?
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
-
-
-
-
?
NATURAL SUBSTRATE
NATURAL 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 UpsiC + FADH2
tetrahydrofolate + tRNA TpsiC + FAD
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
FAD
contains tightly bound flavin
FADH2
NADH
-
is used in the assay as electron donor
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8
-
assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
assay at
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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Uniprot
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
33600
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untagged protein
47900
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His-tagged fusion protein
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C193A
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mutant is nearly as active as the wild-type enzyme
C226A
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mutant loses both the tRNA methylation activity and the capacity to form a covalent complex with the 5-FU-mini-RNA
C53A
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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
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mutant is nearly as active as the wild-type enzyme
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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using Ni-NTA chromatography
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
expressed in Escherichia coli as a His-tagged fusion protein
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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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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Delk, A.S.; Romeo, J.M.; Nagle, D.P.; Rabinowitz, J.C.
Biosynthesis of ribothymidine in the transfer RNA of Streptococcus faecalis and Bacillus subtilis. A methylation of RNA involving 5,10-methylenetetrahydrofolate
J. Biol. Chem.
251
7649-7656
1976
Bacillus subtilis, Enterococcus faecalis, no activity in Micrococcus luteus
Manually annotated by BRENDA team
Delk, A.S.; Nagle, D.P.; Rabinowitz, J.C.
The methylenetetrahydrofolate-mediated biosynthesis of ribothymidine in the transfer-RNA of Streptococcus faecalis: incorporation of hydrogen from solvent into the methyl moiety
Biochem. Biophys. Res. Commun.
86
244-251
1979
Bacillus cereus, Bacillus subtilis, Enterococcus faecalis
Manually annotated by BRENDA team
Delk, A.S.; Nagle, D.P.; Rabinowitz, J.C.
Methylenetetrahydrofolate-dependent biosynthesis of ribothymidine in transfer RNA of Streptococcus faecalis. Evidence for reduction of the 1-carbon unit by FADH2
J. Biol. Chem.
255
4387-4390
1980
Bacillus subtilis, Enterococcus faecalis
Manually annotated by BRENDA team
Urbonavicius, J.; Skouloubris, S.; Myllykallio, H.; Grosjean, H.
Identification of a novel gene encoding a flavin-dependent tRNA:m5U methyltransferase in bacteria -evolutionary implications
Nucleic Acids Res.
33
3955-3964
2005
Bacillus subtilis (P39815), Bacillus subtilis
Manually annotated by BRENDA team
Hamdane, D.; Guerineau, V.; Un, S.; Golinelli-Pimpaneau, B.
A catalytic intermediate and several flavin redox states stabilized by folate-dependent tRNA methyltransferase from Bacillus subtilis
Biochemistry
50
5208-5219
2011
Bacillus subtilis
Manually annotated by BRENDA team
Hamdane, D.; Argentini, M.; Cornu, D.; Myllykallio, H.; Skouloubris, S.; Hui-Bon-Hoa, G.; Golinelli-Pimpaneau, B.
Insights into folate/FAD-dependent tRNA methyltransferase mechanism: role of two highly conserved cysteines in catalysis
J. Biol. Chem.
286
36268-36280
2011
Bacillus subtilis
Manually annotated by BRENDA team
Hamdane, D.; Skouloubris, S.; Myllykallio, H.; Golinelli-Pimpaneau, B.
Expression and purification of untagged and histidine-tagged folate-dependent tRNA:m5U54 methyltransferase from Bacillus subtilis
Protein Expr. Purif.
73
83-89
2010
Bacillus subtilis
Manually annotated by BRENDA team