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Information on EC 2.1.1.200 - tRNA (cytidine32/uridine32-2'-O)-methyltransferase and Organism(s) Escherichia coli and UniProt Accession P0AE01

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IUBMB Comments
In Escherichia coli YfhQ is the only methyltransferase responsible for the formation of 2'-O-methylcytidine32 in tRNA. No methylation of cytosine34 in tRNALeu(CAA). In vitro the enzyme 2-O-methylates cytidine32 of tRNASer1 and uridine32 of tRNAGln2.
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This record set is specific for:
Escherichia coli
UNIPROT: P0AE01
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The taxonomic range for the selected organisms is: Escherichia coli
The expected taxonomic range for this enzyme is: Bacteria, Archaea
Synonyms
trmet(xm32), trna:cm32/um32 methyltransferase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tRNA:Cm32/Um32 methyltransferase
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SYSTEMATIC NAME
IUBMB Comments
S-adenosyl-L-methionine:tRNA (cytidine32/uridine32-2'-O)-methyltransferase
In Escherichia coli YfhQ is the only methyltransferase responsible for the formation of 2'-O-methylcytidine32 in tRNA. No methylation of cytosine34 in tRNALeu(CAA). In vitro the enzyme 2-O-methylates cytidine32 of tRNASer1 and uridine32 of tRNAGln2.
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 + cytidine32 in tRNA
S-adenosyl-L-homocysteine + 2'-O-methylcytidine32 in tRNA
show the reaction diagram
S-adenosyl-L-methionine + nucleoside32 in EctRNAfMet1(CAU)
S-adenosyl-L-homocysteine + 2'-O-methylnucleoside32 in EctRNAfMet1(CAU)
show the reaction diagram
-
-
-
?
S-adenosyl-L-methionine + nucleoside32 in EctRNAfMet2(CAU)
S-adenosyl-L-homocysteine + 2'-O-methylnucleoside32 in EctRNAfMet2(CAU)
show the reaction diagram
-
-
-
?
S-adenosyl-L-methionine + nucleoside32 in EctRNAGln1(UUG)
S-adenosyl-L-homocysteine + 2'-O-methylnucleoside32 in EctRNAGln1(UUG)
show the reaction diagram
-
-
-
?
S-adenosyl-L-methionine + nucleoside32 in EctRNAGln2(CUG)
S-adenosyl-L-homocysteine + 2'-O-methylnucleoside32 in EctRNAGln2(CUG)
show the reaction diagram
-
-
-
?
S-adenosyl-L-methionine + nucleoside32 in EctRNASer1(UGA)
S-adenosyl-L-homocysteine + 2'-O-methylnucleoside32 in EctRNASer1(UGA)
show the reaction diagram
-
-
-
?
S-adenosyl-L-methionine + nucleoside32 in EctRNATrp1(CCA)
S-adenosyl-L-homocysteine + 2'-O-methylnucleoside32 in EctRNATrp1(CCA)
show the reaction diagram
-
-
-
?
S-adenosyl-L-methionine + nucleoside32 in tRNA
S-adenosyl-L-homocysteine + 2'-O-methylnucleoside32 in tRNA
show the reaction diagram
-
-
-
?
S-adenosyl-L-methionine + uridine32 in tRNA
S-adenosyl-L-homocysteine + 2'-O-methyluridine32 in tRNA
show the reaction diagram
additional information
?
-
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
S-adenosyl-L-methionine + cytidine32 in tRNA
S-adenosyl-L-homocysteine + 2'-O-methylcytidine32 in tRNA
show the reaction diagram
presence of 2'-O-methylated cytidine at position 32 in tRNAfMet1(CAU), tRNAfMet2(CAU), tRNASer1(UGA), and tRNATrp1(CCA). In Escherichia coli YfhQ is the only methyltransferase responsible for the formation of Cm32 in tRNA
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-
?
S-adenosyl-L-methionine + nucleoside32 in tRNA
S-adenosyl-L-homocysteine + 2'-O-methylnucleoside32 in tRNA
show the reaction diagram
-
-
-
?
S-adenosyl-L-methionine + uridine32 in tRNA
S-adenosyl-L-homocysteine + 2'-O-methyluridine32 in tRNA
show the reaction diagram
presence of 2'-O-methylated uridine in position 32 of the anticodon loop in tRNAGln1(UUG) and tRNAGln2(CUG)
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-
?
additional information
?
-
bacterial TrmJs recognize substrate tRNAs and specifically catalyze a 2'-O modification at ribose 32. All six Escherichia coli tRNAs with 2'-O-methylated nucleosides at position 32 are substrates of EcTrmJ. The elbow region of tRNA, but not the amino acid acceptor stem, is needed for the methylation reaction. tRNA recognition by EcTrmJ involves the cooperative influences of conserved residues from both the SPOUT and extensional domains, and this process is regulated by the flexible hinge region that connects these two domains
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-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
S-adenosyl-L-methionine
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00067
nucleoside32 in EctRNAfMet1(CAU)
pH 9.0, 37°C, recombinant wild-type enzyme
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0.00081
nucleoside32 in EctRNAfMet2(CAU)
pH 9.0, 37°C, recombinant wild-type enzyme
-
0.00988
nucleoside32 in EctRNAGln1(UUG)
pH 9.0, 37°C, recombinant wild-type enzyme
-
0.00579
nucleoside32 in EctRNAGln2(CUG)
pH 9.0, 37°C, recombinant wild-type enzyme
-
0.01182
nucleoside32 in EctRNASer1(UGA)
pH 9.0, 37°C, recombinant wild-type enzyme
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0.00094
nucleoside32 in EctRNATrp1(CCA)
pH 9.0, 37°C, recombinant wild-type enzyme
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0253
nucleoside32 in EctRNAfMet1(CAU)
pH 9.0, 37°C, recombinant wild-type enzyme
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0.0237
nucleoside32 in EctRNAfMet2(CAU)
pH 9.0, 37°C, recombinant wild-type enzyme
-
0.02
nucleoside32 in EctRNAGln1(UUG)
pH 9.0, 37°C, recombinant wild-type enzyme
-
0.0235
nucleoside32 in EctRNAGln2(CUG)
pH 9.0, 37°C, recombinant wild-type enzyme
-
0.0383
nucleoside32 in EctRNASer1(UGA)
pH 9.0, 37°C, recombinant wild-type enzyme
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0.0218
nucleoside32 in EctRNATrp1(CCA)
pH 9.0, 37°C, recombinant wild-type enzyme
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
TrmJ proteins from the SPOUT methyltransferase superfamily are tRNA Xm32 modification enzymes that occur in bacteria and archaea. Unlike archaeal TrmJ, bacterial TrmJ require full-length tRNA molecules as substrates
physiological function
methylation of ribose moieties in tRNA is frequent, especially at position 32 where it is commonplace in all three domains of life
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
29168
2 * 29168, calculated from sequence
30000
2 * 30000, SDS-PAGE in presence of 7.5% 2-mercaptoethanol
58000
gel filtration
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
additional information
the dimerization of C-terminal domain is important for the catalytic activity of EcTrmJ
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified recombinant C-terminally His6-tagged wild-type enzyme, hanging drop vapour diffusion method, mixing of 10 mg/ml of protein 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl2, and 2 mM S-adenosyl-L-homocysteine, with 3.6 M NaCl and 0.1 M HEPES, pH 8.2, 20°C, 2 months, X-ray diffraction structure determination and analysis, full-length EcTrmJ forms an unusual dimer in the asymmetric unit, with both the catalytic SPOUT domain and C-terminal extension forming separate dimeric associations in the crystal structure, molecular replacement
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E225A
site-directed mutagenesis, inactive mutant
F199A
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
G231A
site-directed mutagenesis, inactive mutant
I228A
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
L229A
site-directed mutagenesis, inactive mutant
additional information
generation of several partial deletion mutants of TrmJ, including single site mutants, DELTA2 (deleted residues 171-172), DELTA4 (deleted residues 171-174), DELTA7 (deleted residues 169-175), DELTA10 (deleted residues 167-176), DELTA12 (deleted residues 166-177), NTD (residues 1-170) and CTD (residues 171-246)
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant C-terminally His6-tagged wild-type enzyme and deletion mutants from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant expression of C-terminally His6-tagged wild-type enzyme and deletion mutants in Escherichia coli strain BL21(DE3)
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Purta, E.; van Vliet, F.; Tkaczuk, K.L.; Dunin-Horkawicz, S.; Mori, H.; Droogmans, L.; Bujnicki, J.M.
The yfhQ gene of Escherichia coli encodes a tRNA:Cm32/Um32 methyltransferase
BMC Mol. Biol.
7
23
2006
Escherichia coli (P0AE01), Escherichia coli
Manually annotated by BRENDA team
Liu, R.J.; Long, T.; Zhou, M.; Zhou, X.L.; Wang, E.D.
tRNA recognition by a bacterial tRNA Xm32 modification enzyme from the SPOUT methyltransferase superfamily
Nucleic Acids Res.
43
7489-7503
2015
Escherichia coli (P0AE01), Escherichia coli K-12 MG1655 (P0AE01)
Manually annotated by BRENDA team