|Gene:||mnmC||Accession Numbers: G7199 (EcoCyc), b2324, ECK2318|
Synonyms: yfcK, trmC
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 [Kim13d].
[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.
|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
|Biological Process:||GO:0002098 - tRNA wobble uridine modification
GO:0030488 - tRNA methylation [Bjork78]
GO:0002097 - tRNA wobble base modification [GOA06]
GO:0008033 - tRNA processing [UniProtGOA11a, GOA01a]
GO:0008152 - metabolic process [UniProtGOA11a]
GO:0032259 - methylation [UniProtGOA11a]
GO:0055114 - oxidation-reduction process [UniProtGOA11a, GOA01a]
|Molecular Function:||GO:0004808 - tRNA (5-methylaminomethyl-2-thiouridylate)-methyltransferase activity
[GOA01, GOA01a, Hagervall87]
GO:0071949 - FAD binding [Roovers08]
GO:0003824 - catalytic activity [UniProtGOA11a]
GO:0008168 - methyltransferase activity [UniProtGOA11a, GOA01a]
GO:0008757 - S-adenosylmethionine-dependent methyltransferase activity [GOA06]
GO:0016491 - oxidoreductase activity [UniProtGOA11a, GOA06, GOA01a]
GO:0016645 - oxidoreductase activity, acting on the CH-NH group of donors [GOA01a]
GO:0016740 - transferase activity [UniProtGOA11a]
GO:0050660 - flavin adenine dinucleotide binding [GOA06]
|Cellular Component:||GO:0005737 - cytoplasm
[UniProtGOA11, UniProtGOA11a, GOA06]
GO:0005829 - cytosol [DiazMejia09]
|MultiFun Terms:||information transfer → RNA related → RNA modification|
|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]|
Enzymatic reaction of: 5-aminomethyl-2-thiouridine synthase (fused 5-methylaminomethyl-2-thiouridine-forming methyltransferase and FAD-dependent demodification enzyme)
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].
pH(opt): 8-8.5 [Hagervall87]
Enzymatic reaction of: 5-methylaminomethyl-2-thiouridine-forming methyltransferase
EC Number: 22.214.171.124
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.
pH(opt): 8-8.5 [Hagervall87]
|Protein-Segment||1 -> 245|
|Protein-Segment||270 -> 668|
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). "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
Bjork78a: 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
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
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
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
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
©2014 SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025-3493