Escherichia coli K-12 substr. MG1655 Enzyme: fused 5-methylaminomethyl-2-thiouridine-forming methyltransferase and FAD-dependent demodification enzyme

Gene: mnmC Accession Numbers: G7199 (EcoCyc), b2324, ECK2318

Synonyms: yfcK, trmC

Regulation Summary Diagram: ?

Regulation summary diagram for mnmC

The tRNAs specific for glutamate, lysine, and possibly glutamine contain the hypermodified nucleoside 5-methylaminomethyl-2-thiouridine (mnm5s2U) in position 34, the wobble position. The MnmC protein was shown to catalyze the formation of mnm5s2U from 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U) in tRNA [Hagervall87, Bujnicki04]. Both the tRNA species and growth conditions modulate synthesis of the wobble base modifications [Moukadiri14].

A tentative reaction mechanism has been proposed. The enzyme catalyzes two steps, an initial FAD-dependent demodification of cmnm5s2U to nm5s2U followed by the transfer of a methyl group from AdoMet to nm5s2U to produce mnm5s2U [Hagervall87, Bujnicki04]. The second reaction occurs faster than the first reaction, indicating that the enzyme is tuned to produce fully mnm5s2U-modified tRNA without accumulating the nm5s2U-modified tRNA intermediate [Pearson11]. tRNAGlncmnm5s2UUG and tRNALeucmnm5UmAA are not substrates for the demodification activity of MnmC [Moukadiri14].

The MnmC protein consists of two conserved domains, an N-terminal methyltransferase domain and a C-terminal FAD-dependent oxidoreductase domain. The purified recombinant protein contains a flavin derivative [Bujnicki04]. As already indicated by the mutant phenotypes of the trmC1 and trmC2 mutant alleles [Hagervall84], a structural model of MnmC suggests that the initial demodification reaction is catalyzed by the C-terminal domain of the protein, while the methylation reaction is catalyzed by the N-terminal domain. Site-directed mutagenesis of predicted active site residues and separate expression of the two domains confirmed this hypothesis [Roovers08, Moukadiri14].

A crystal structure of MnmC containing the FAD cofactor has been solved at 3 Å resolution. The catalytic sites of the two domains face opposite sides of the protein, arguing against substrate channeling [Kitamura11]. A crystal structure of the E. coli B enzyme (which is 99% identical to the K-12 enzyme) containing both FAD and SAM has also been solved [Kim13a].

[Pearson11] report that an mnmC null mutant has a lower growth rate than wild type, while [Moukadiri14] found no effect of any tested mnmC mutation on growth rate, although growth competition assays showed that the wild type does outcompete certain mnmC mutants.

Reviews: [Armengod12, El12]

Citations: [Bjork78]

Locations: cytosol

Map Position: [2,439,786 -> 2,441,792] (52.59 centisomes, 189°)
Length: 2007 bp / 668 aa

Molecular Weight of Polypeptide: 74.434 kD (from nucleotide sequence), 79 kD (experimental) [Hagervall87 ]

Unification Links: ASAP:ABE-0007681 , DIP:DIP-28058N , EchoBASE:EB3867 , EcoGene:EG14114 , EcoliWiki:b2324 , ModBase:P77182 , OU-Microarray:b2324 , PortEco:mnmC , PR:PRO_000023255 , Pride:P77182 , Protein Model Portal:P77182 , RefSeq:NP_416827 , RegulonDB:G7199 , SMR:P77182 , String:511145.b2324 , UniProt:P77182

Relationship Links: InterPro:IN-FAMILY:IPR006076 , InterPro:IN-FAMILY:IPR008471 , InterPro:IN-FAMILY:IPR017610 , InterPro:IN-FAMILY:IPR023032 , InterPro:IN-FAMILY:IPR029063 , PDB:Structure:3AWI , Pfam:IN-FAMILY:PF01266 , Pfam:IN-FAMILY:PF05430

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

GO Terms:

Biological Process: GO:0002098 - tRNA wobble uridine modification Inferred from experiment [Pearson11]
GO:0030488 - tRNA methylation Inferred from experiment [Bjork78a]
GO:0002097 - tRNA wobble base modification Inferred by computational analysis [GOA06]
GO:0008033 - tRNA processing Inferred by computational analysis [UniProtGOA11a, GOA01a]
GO:0008152 - metabolic process Inferred by computational analysis [UniProtGOA11a]
GO:0032259 - methylation Inferred by computational analysis [UniProtGOA11a]
GO:0055114 - oxidation-reduction process Inferred by computational analysis [UniProtGOA11a, GOA01a]
Molecular Function: GO:0004808 - tRNA (5-methylaminomethyl-2-thiouridylate)-methyltransferase activity Inferred from experiment Inferred by computational analysis [GOA01, GOA01a, Hagervall87]
GO:0071949 - FAD binding Inferred from experiment [Roovers08]
GO:0003824 - catalytic activity Inferred by computational analysis [UniProtGOA11a]
GO:0008168 - methyltransferase activity Inferred by computational analysis [UniProtGOA11a, GOA01a]
GO:0008757 - S-adenosylmethionine-dependent methyltransferase activity Inferred by computational analysis [GOA06]
GO:0016491 - oxidoreductase activity Inferred by computational analysis [UniProtGOA11a, GOA06, GOA01a]
GO:0016645 - oxidoreductase activity, acting on the CH-NH group of donors Inferred by computational analysis [GOA01a]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11a]
GO:0050660 - flavin adenine dinucleotide binding Inferred by computational analysis [GOA06]
Cellular Component: GO:0005737 - cytoplasm Inferred by computational analysis [UniProtGOA11, UniProtGOA11a, GOA06]
GO:0005829 - cytosol Inferred by computational analysis [DiazMejia09]

MultiFun Terms: information transfer RNA related RNA modification

Essentiality data for mnmC knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enriched Yes 37 Aerobic 6.95   Yes [Gerdes03, Comment 1]
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]

Last-Curated ? 13-Dec-2013 by Keseler I , SRI International

Enzymatic reaction of: 5-aminomethyl-2-thiouridine synthase (fused 5-methylaminomethyl-2-thiouridine-forming methyltransferase and FAD-dependent demodification enzyme)

a 5-carboxymethylaminomethyl-2-thiouridine in tRNA <=> a 5-aminomethyl-2-thiouridine in tRNA + glyoxylate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is physiologically favored in the direction shown.

A tentative reaction mechanism, including production of glyoxylate, was proposed by [Bujnicki04].

Inhibitors (Unknown Mechanism): Mg2+ [Hagervall87]

Kinetic Parameters:

Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
a 5-carboxymethylaminomethyl-2-thiouridine in tRNA

pH(opt): 8-8.5 [Hagervall87]

Enzymatic reaction of: 5-methylaminomethyl-2-thiouridine-forming methyltransferase

Synonyms: TrMnmMet(mnm5{s2}U34)

EC Number:

a 5-aminomethyl-2-thiouridine in tRNA + S-adenosyl-L-methionine <=> S-adenosyl-L-homocysteine + a 5-methylaminomethyl-2-thiouridine in tRNA + H+

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

The reaction is physiologically favored in the direction shown.

Activators (Unknown Mechanism): ammonium [Hagervall87]

Inhibitors (Unknown Mechanism): Mg2+ [Hagervall87]

Kinetic Parameters:

Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
a 5-aminomethyl-2-thiouridine in tRNA

pH(opt): 8-8.5 [Hagervall87]

Sequence Features

Protein sequence of fused 5-methylaminomethyl-2-thiouridine-forming methyltransferase and FAD-dependent demodification enzyme with features indicated

Feature Class Location Citations Comment
Protein-Segment 1 -> 245
UniProt: tRNA (mnm(5)s(2)U34)-methyltransferase; Sequence Annotation Type: region of interest;
Mutagenesis-Variant 64
[Roovers08, UniProt11]
UniProt: Loss of methyltransferase activity.
Mutagenesis-Variant 178
[Roovers08, UniProt11]
UniProt: Strong decrease in methyltransferase activity.
Mutagenesis-Variant 180
[Roovers08, UniProt11]
UniProt: Strong decrease in methyltransferase activity.
Protein-Segment 270 -> 668
UniProt: FAD-dependent cmnm(5)s(2)U34 oxidoreductase; Sequence Annotation Type: region of interest;
Mutagenesis-Variant 271
[Roovers08, UniProt11]
UniProt: 4-fold decrease in activity, but no change in FAD binding.
Mutagenesis-Variant 567
[Roovers08, UniProt11]
UniProt: Loss of activity, but no change in FAD binding.
Mutagenesis-Variant 618
[Roovers08, UniProt11]
UniProt: Loss of activity, but no change in FAD binding.

Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Unit:

Transcription-unit diagram


Peter D. Karp on Wed Jan 18, 2006:
Gene left-end position adjusted based on analysis performed in the 2005 E. coli annotation update [Riley06 ].
Markus Krummenacker on Tue Oct 14, 1997:
Gene object created from Blattner lab Genbank (v. M52) entry.


Armengod12: Armengod ME, Moukadiri I, Prado S, Ruiz-Partida R, Benitez-Paez A, Villarroya M, Lomas R, Garzon MJ, Martinez-Zamora A, Meseguer S, Navarro-Gonzalez C (2012). "Enzymology of tRNA modification in the bacterial MnmEG pathway." Biochimie 94(7);1510-20. PMID: 22386868

Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554

Bjork78: Bjork GR, Kjellin-Straby K (1978). "General screening procedure for RNA modificationless mutants: isolation of Escherichia coli strains with specific defects in RNA methylation." J Bacteriol 133(2);499-507. PMID: 342494

Bjork78a: Bjork GR, Kjellin-Straby K (1978). "Escherichia coli mutants with defects in the biosynthesis of 5-methylaminomethyl-2-thio-uridine or 1-methylguanosine in their tRNA." J Bacteriol 133(2);508-17. PMID: 342495

Bujnicki04: Bujnicki JM, Oudjama Y, Roovers M, Owczarek S, Caillet J, Droogmans L (2004). "Identification of a bifunctional enzyme MnmC involved in the biosynthesis of a hypermodified uridine in the wobble position of tRNA." RNA 10(8);1236-42. PMID: 15247431

DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114

El12: El Yacoubi B, Bailly M, de Crecy-Lagard V (2012). "Biosynthesis and function of posttranscriptional modifications of transfer RNAs." Annu Rev Genet 46;69-95. PMID: 22905870

Gerdes03: Gerdes SY, Scholle MD, Campbell JW, Balazsi G, Ravasz E, Daugherty MD, Somera AL, Kyrpides NC, Anderson I, Gelfand MS, Bhattacharya A, Kapatral V, D'Souza M, Baev MV, Grechkin Y, Mseeh F, Fonstein MY, Overbeek R, Barabasi AL, Oltvai ZN, Osterman AL (2003). "Experimental determination and system level analysis of essential genes in Escherichia coli MG1655." J Bacteriol 185(19);5673-84. PMID: 13129938

GOA01: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

GOA01a: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA06: GOA, SIB (2006). "Electronic Gene Ontology annotations created by transferring manual GO annotations between orthologous microbial proteins."

Hagervall84: Hagervall TG, Bjork GR (1984). "Undermodification in the first position of the anticodon of supG-tRNA reduces translational efficiency." Mol Gen Genet 196(2);194-200. PMID: 6387394

Hagervall87: Hagervall TG, Edmonds CG, McCloskey JA, Bjork GR (1987). "Transfer RNA(5-methylaminomethyl-2-thiouridine)-methyltransferase from Escherichia coli K-12 has two enzymatic activities." J Biol Chem 262(18);8488-95. PMID: 3298234

Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394

Kim13a: Kim J, Almo SC (2013). "Structural basis for hypermodification of the wobble uridine in tRNA by bifunctional enzyme MnmC." BMC Struct Biol 13;5. PMID: 23617613

Kitamura11: Kitamura A, Sengoku T, Nishimoto M, Yokoyama S, Bessho Y (2011). "Crystal structure of the bifunctional tRNA modification enzyme MnmC from Escherichia coli." Protein Sci 20(7);1105-13. PMID: 21574198

Moukadiri14: Moukadiri I, Garzon MJ, Bjork GR, Armengod ME (2014). "The output of the tRNA modification pathways controlled by the Escherichia coli MnmEG and MnmC enzymes depends on the growth conditions and the tRNA species." Nucleic Acids Res 42(4);2602-23. PMID: 24293650

Pearson11: Pearson D, Carell T (2011). "Assay of both activities of the bifunctional tRNA-modifying enzyme MnmC reveals a kinetic basis for selective full modification of cmnm5s2U to mnm5s2U." Nucleic Acids Res 39(11);4818-26. PMID: 21306992

Riley06: Riley M, Abe T, Arnaud MB, Berlyn MK, Blattner FR, Chaudhuri RR, Glasner JD, Horiuchi T, Keseler IM, Kosuge T, Mori H, Perna NT, Plunkett G, Rudd KE, Serres MH, Thomas GH, Thomson NR, Wishart D, Wanner BL (2006). "Escherichia coli K-12: a cooperatively developed annotation snapshot--2005." Nucleic Acids Res 34(1);1-9. PMID: 16397293

Roovers08: Roovers M, Oudjama Y, Kaminska KH, Purta E, Caillet J, Droogmans L, Bujnicki JM (2008). "Sequence-structure-function analysis of the bifunctional enzyme MnmC that catalyses the last two steps in the biosynthesis of hypermodified nucleoside mnm5s2U in tRNA." Proteins 71(4);2076-85. PMID: 18186482

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

UniProt11: UniProt Consortium (2011). "UniProt version 2011-06 released on 2011-06-30 00:00:00." Database.

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on the manual assignment of UniProtKB Subcellular Location terms in UniProtKB/Swiss-Prot entries."

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
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