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Information on EC 2.1.1.229 - tRNA (carboxymethyluridine34-5-O)-methyltransferase and Organism(s) Escherichia coli and UniProt Accession P76290
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2.1.1.229 tRNA (carboxymethyluridine34-5-O)-methyltransferaseIUBMB Comments
The enzyme catalyses the posttranslational modification of uridine residues at the wobble position 34 of the anticodon loop of tRNA.
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Word Map
- 2.1.1.229
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wobble
- methyltransferases
-
5-methoxycarbonylmethyluridine
- ribonucleotide
-
anticodon
-
gene-specific
- trnagluuuc
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translationally
- medicine
The taxonomic range for the selected organisms is: Escherichia coli
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea
Reaction Schemes
+
=
+
+
carboxymethyluridine34 in tRNA
=
+
5-(2-methoxy-2-oxoethyl)uridine34 in tRNA
Synonyms
trna methyltransferase 9, trm9p, trm9-trm112, yml014w, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
MnmC
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
S-adenosyl-L-methionine + carboxymethyluridine34 in tRNA = S-adenosyl-L-homocysteine + 5-(2-methoxy-2-oxoethyl)uridine34 in tRNA
S-adenosyl-L-methionine + carboxymethyluridine34 in tRNA = S-adenosyl-L-homocysteine + 5-(2-methoxy-2-oxoethyl)uridine34 in tRNA
aspects of substrate recognition and catalytic mechanism. overview
S-adenosyl-L-methionine + carboxymethyluridine34 in tRNA = S-adenosyl-L-homocysteine + 5-(2-methoxy-2-oxoethyl)uridine34 in tRNA
the C-terminal domain catalyzes the FAD-dependent oxidation of the Calpha-N bond in carboxymethylaminomethyl uridine34. The resulting imine intermediate is (presumably) non-enzymatically hydrolyzes to 5-aminomethyl uridine, followed by S-adenosyl-L-methionine-dependent methylation to yield methylaminomethyl uridine34 in the N-terminal domain active site
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PATHWAY SOURCE
PATHWAYS
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-, -
SYSTEMATIC NAME
IUBMB Comments
S-adenosyl-L-methionine:tRNA (carboxymethyluridine34-5-O)-methyltransferase
The enzyme catalyses the posttranslational modification of uridine residues at the wobble position 34 of the anticodon loop of tRNA.
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
S-adenosyl-S-carboxymethyl-L-homocysteine + 5-methoxyuridine34 in tRNA
S-adenosyl-L-methionine + uridine 5-oxyacetic acid in tRNA
proposed modification pathway of 5-oxyuridine derivatives, overview
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-
?
S-adenosyl-L-methionine + carboxymethylaminomethyl 2-thiouridine34 in tRNAGlu
S-adenosyl-L-homocysteine + methylaminomethyl 2-thiouridine34 in tRNAGlu + hydroxyacetate
-
-
-
-
?
S-adenosyl-L-methionine + carboxymethylaminomethyl 2-thiouridine34 in tRNALys
S-adenosyl-L-homocysteine + methylaminomethyl 2-thiouridine34 in tRNALys + hydroxyacetate
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-
-
-
?
S-adenosyl-L-methionine + carboxymethylaminomethyl uridine34 in tRNAArg
S-adenosyl-L-homocysteine + methylaminomethyl uridine34 in tRNAArg + hydroxyacetate
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-
-
-
?
S-adenosyl-L-methionine + carboxymethyluridine34 in tRNA
S-adenosyl-L-homocysteine + 5-(2-methoxy-2-oxoethyl)uridine34 in tRNA
S-adenosyl-L-methionine + carboxymethyluridine34 in tRNA
S-adenosyl-L-homocysteine + 5-(2-methoxy-2-oxoethyl)uridine34 in tRNA
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-
-
?
S-adenosyl-L-methionine + carboxymethyluridine34 in tRNA
S-adenosyl-L-homocysteine + 5-(2-methoxy-2-oxoethyl)uridine34 in tRNA
residue Arg567 os involved in tRNA substrate recognition
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-
?
additional information
?
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the bifunctional enzyme MnmC catalyzes the two consecutive reactions that convert 5-carboxymethylaminomethyl uridine to 5-methylaminomethyl uridine. The C-terminal domain of MnmC is responsible for the FAD-dependent deacetylation of cmnm5U to 5-aminomethyl uridine, whereas the N-terminal domain catalyzes the subsequent S-adenosyl-L-methionine-dependent methylation of 5-aminomethyl uridine, leading to the final product, 5-methylaminomethyl uridine, coordination of the two consecutive reactions, overview
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-
?
additional information
?
-
-
methylaminomethyl modification of uridine or 2-thiouridine (mnm5U34 or mnm5s2U34) at the wobble position of tRNAs specific for glutamate, lysine and arginine are observed in Escherichia coli and allow for specific recognition of codons ending in A or G. In the biosynthetic pathway responsible for this posttranscriptional modification, the bifunctional enzyme MnmC catalyzes the conversion of its hypermodified substrate carboxymethylaminomethyl uridine (cmnm5U34) to mnm5U34. MnmC catalyzes the FAD-dependent oxidative cleavage of carboxymethyl group from cmnm5U34 via an imine intermediate to generate aminomethyl uridine (nm5U34), which is subsequently methylated by S-adenosyl-L-methionine to yield methylaminomethyl uridine (mnm5U34)
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-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
S-adenosyl-L-methionine + carboxymethylaminomethyl 2-thiouridine34 in tRNAGlu
S-adenosyl-L-homocysteine + methylaminomethyl 2-thiouridine34 in tRNAGlu + hydroxyacetate
-
-
-
-
?
S-adenosyl-L-methionine + carboxymethylaminomethyl 2-thiouridine34 in tRNALys
S-adenosyl-L-homocysteine + methylaminomethyl 2-thiouridine34 in tRNALys + hydroxyacetate
-
-
-
-
?
S-adenosyl-L-methionine + carboxymethylaminomethyl uridine34 in tRNAArg
S-adenosyl-L-homocysteine + methylaminomethyl uridine34 in tRNAArg + hydroxyacetate
-
-
-
-
?
S-adenosyl-L-methionine + carboxymethyluridine34 in tRNA
S-adenosyl-L-homocysteine + 5-(2-methoxy-2-oxoethyl)uridine34 in tRNA
-
-
-
?
additional information
?
-
the bifunctional enzyme MnmC catalyzes the two consecutive reactions that convert 5-carboxymethylaminomethyl uridine to 5-methylaminomethyl uridine. The C-terminal domain of MnmC is responsible for the FAD-dependent deacetylation of cmnm5U to 5-aminomethyl uridine, whereas the N-terminal domain catalyzes the subsequent S-adenosyl-L-methionine-dependent methylation of 5-aminomethyl uridine, leading to the final product, 5-methylaminomethyl uridine, coordination of the two consecutive reactions, overview
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-
?
additional information
?
-
-
methylaminomethyl modification of uridine or 2-thiouridine (mnm5U34 or mnm5s2U34) at the wobble position of tRNAs specific for glutamate, lysine and arginine are observed in Escherichia coli and allow for specific recognition of codons ending in A or G. In the biosynthetic pathway responsible for this posttranscriptional modification, the bifunctional enzyme MnmC catalyzes the conversion of its hypermodified substrate carboxymethylaminomethyl uridine (cmnm5U34) to mnm5U34. MnmC catalyzes the FAD-dependent oxidative cleavage of carboxymethyl group from cmnm5U34 via an imine intermediate to generate aminomethyl uridine (nm5U34), which is subsequently methylated by S-adenosyl-L-methionine to yield methylaminomethyl uridine (mnm5U34)
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-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
S-adenosyl-S-carboxymethyl-L-homocysteine
i.e. [(3S)-3-amino-3-carboxypropyl]{[(2S,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methyl}(carboxymethyl)sulfanium, the enzyme contains a cofactor, S-adenosyl-S-carboxymethyl-L-homocysteine (SCM-SAH), in which the donor methyl group is substituted by a carboxymethyl group. The carboxyl moiety forms a salt-bridge interaction with Arg199 that is conserved in a large group of CmoA-related proteins but is not conserved in other S-adenosyl-L-methionine-containing enzymes. The active site contains one molecule cofactor S-adenosyl-S-carboxymethyl-L-homocysteine per monomer, and not S-adenosyl-L-methionine
FAD
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required for oxidative cleavage of carboxymethyl group from cmnm5U34, FAD-binding site structure, overview
S-adenosyl-L-methionine
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dependent on, the N-terminal MnmC2 domain is composed of residues 1-245 and contains the SAM binding site. The binding pocket is composed of mostly hydrophobic residues, except for Glu101 and Asp178
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
conservation of Arg199, the key residue of CmoA that stabilizes the negative charge of the carboxyl group of the S-adenosyl-S-carboxymethyl-L-homocysteine cofactor, suggests that these proteins contain the S-adenosyl-S-carboxymethyl-L-homocysteine cofactor instead of S-adenosyl-L-methionine. The equivalent residue in known S-adenosyl-L-methionine-dependent methyltransferases is not conserved
physiological function
uridine at position 34 of bacterial transfer RNAs is commonly modified to uridine-5-oxyacetic acid (cmo5U) to increase the decoding capacity. The protein CmoA is involved in the formation of cmo5U
evolution
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comparison of the MnmC2 active sites between Escherichia coli MnmC and Yersinia pestis MnmC, overview. Structural comparison with MnmC2 of Aquifex aeolicus
metabolism
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MnmC (formally known as YfcK or TrmC) is a bifunctional enzyme responsible for the final two steps of biosynthetic pathway of mnm5s2U in tRNAGlu and tRNALys, and mnm5U in tRNAArg
physiological function
posttranscriptional modifications of bases within the tRNA anticodon significantly affect the decoding system, uridines at the wobble position U34 of some tRNAs are modified to 5-methyluridine derivatives. These xm5U34-containing tRNAs read codons ending with A or G, whereas tRNAs with the unmodified U34 are able to read all four synonymous codons of a family box
additional information
-
crystal structure of MnmC from the Gram negative bacterium reveals the overall architecture of the enzyme and the relative disposition of the two independent catalytic domains: a Rossmann-fold domain containing the S-adenosyl-L-methionine binding site and an FAD containing domain structurally homologous to glycine oxidase from Bacillus subtilis. The structure of MnmC also reveals the detailed atomic interactions at the interdomain interface and provide spatial restraints relevant to the overall catalytic mechanism
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
55600
2 x 52500, gel filtration, mass spectrometry, and crystal structure analysis, 2 * 55600, about, sequence calculation. There are two molecules of CmoA present in the asymmetric unit, both molecules of CmoA contain the novel derivative S-adenosyl-S-carboxymethyl-L-homocysteine, the two molecules adopt the same conformation
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
2 x 52500, gel filtration, mass spectrometry, and crystal structure analysis, 2 * 55600, about, sequence calculation. There are two molecules of CmoA present in the asymmetric unit, both molecules of CmoA contain the novel derivative S-adenosyl-S-carboxymethyl-L-homocysteine, the two molecules adopt the same conformation
additional information
methyltransferase domain MnmC2 domain adopts the canonical class I methyltransferase fold
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified recombinant detagged enzyme, sitting drop vapour diffusion method, mixing of 100 nl of 20 mg/ml protein in 200 mM NaCl, and 20 mM Tris, pH 7.5, with 100 nl of precipitation solution containing 0.3 M diethylene glycol, 0.3 M triethylene glycol, 0.3 M tetraethylene glycol, 0.3 M pentaethylene glycol, 0.1 M MOPS/HEPES-Na, pH 7.5, 12.5% w/v PEG 1000, 12.5% w/v PEG 3350, 12.5% w/v MPD, 5 h, X-ray diffraction structure determination and analysis at 1.73 A resolution, molecular replacement using the structure of Haemophilus influenzae YecO, PDB ID 1im8, chain B
purified enzyme in complex with cofactors S-adenosyl-L-methionine and FAD, sitting drop vapor diffusion method, 21°C, by mixing 0.001 ml of 10 mg/ml protein with 0.001 ml of reservoir solution containing 1.8 M tri-ammonium citrate, pH 7.0 and 0.5% ethyl acetate, X-ray diffraction structure determination and analysis at 2.3 A resolution
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purified MnmC containing FAD, sitting drop method, the optimal reservoir solution contains 100 mM Bis-Tris, pH 5.5, containing 25% w/v PEG3350, 250 mM ammonium sulfate, and 10 mM hexamine cobalt(III) chloride, 5 days, X-ray diffraction structure determination and analysis at 3.0 A resolution
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged enzyme from Escherichia coli strain Rosetta pLysS (DE3) by nickel affinity chromatography and gel filtration, followed by cleavage of the N-terminal His6-tag with rhinovirus 3C protease and another step of nickel affinity chromatography to remove the tag
recombinant N-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, tag cleavage with thrombin , and again nickel adffinity chromatography for tag removal, followed by gel filtration
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recombinant wild-type and selenomethinone-labeled enzymes from Escherichia coli by ultracentrifugation, anion exchange and hydrophobic interaction chromatography, another step of anion exchange chromatography and gel filtration
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene cmoA, recombinant expression of N-terminally His-tagged enzyme in Escherichia coli strain Rosetta pLysS (DE3)
gene JW5380, expression of wild-type and selenomethinone-labeled MnmC in Escherichia coli strains Rosetta2(DE3) and B834(DE3), respectively
gene mnmC, recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3)
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Kitamura, A.; Sengoku, T.; Nishimoto, M.; Yokoyama, S.; Bessho, Y.
Crystal structure of the bifunctional tRNA modification enzyme MnmC from Escherichia coli
Protein Sci.
20
1105-1113
2011
Escherichia coli (P77182)
Byrne, R.T.; Whelan, F.; Aller, P.; Bird, L.E.; Dowle, A.; Lobley, C.M.; Reddivari, Y.; Nettleship, J.E.; Owens, R.J.; Antson, A.A.; Waterman, D.G.
S-Adenosyl-S-carboxymethyl-L-homocysteine: a novel cofactor found in the putative tRNA-modifying enzyme CmoA
Acta Crystallogr. Sect. D
69
1090-1098
2013
Escherichia coli (P76290)
EXTERNAL LINKS (specific for EC-Number 2.1.1.229)
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