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Escherichia coli K-12 substr. MG1655 Enzyme: tRNA-guanine transglycosylase



Gene: tgt Accession Numbers: EG10996 (EcoCyc), b0406, ECK0400

Synonyms: vacC(S.f.)

Regulation Summary Diagram: ?

Subunit composition of tRNA-guanine transglycosylase = [Tgt]3
         tRNA-guanine transglycosylase monomer = Tgt

Summary:
tRNA-guanine transglycosylase catalyzes the post-transcriptional base exchange reaction involved in the incorporation of the modified base queuine at the wobble position 34 of tRNAs with GUN anticodons (Asp, Asn, His and Tyr).

The enzyme can form a homohexamer, a dimer of Tgt homotrimers [Garcia93a]. Homotrimerization exhibits concentration dependence [Reuter95].

The catalytic mechanism of the enzyme has been investigated. The enzyme most likely follows a ping-pong mechanism [GoodenoughLashu03]. The reaction proceeds via a covalent Tgt-tRNA intermediate; dissociation of the product RNA is the overall rate-limiting step [Garcia09]. Asp264 appears to be involved in protonation of the leaving guanine and deprotonation of the incoming preQ1 [Kittendorf03]. Asp143 is involved in recognition of guanine in the tRNA substrate [Todorov05, Todorov06]. Other residues shown to be involved in catalytic activity by site-directed mutagenesis are S90 [Reuter94], C265 [Garcia97], and D89 [Kittendorf01]. C145 may have evolved in the eubacterial enzyme to specifically recognize preQ1 rather than the eukaryotic substrate queuine [Chen11d].

Reports have addressed recognition of the tRNA substrate by the enzyme [Curnow93, Nakanishi94, Curnow94, Mueller95, Chong95, Curnow95, Kung98, Kung00, Nonekowski01, Nonekowski02]. Residues C302, C304, C307, and H317 coordinate a zinc, which is important for homotrimerization [Chong95, Garcia96a] and substrate recognition [Chong95]. Additional tRNA modifications are not required for recognition of the tRNA by the enzyme [Curnow93]. The enzyme recognized the anticodon arm region of the tRNA substrate, and is active toward either tRNA monomers or dimers [Curnow94]. Tgt and QueA each recognize the anticodon region of the tRNA substrate [Mueller95]. The enzyme shows activity toward 2'-deoxyuridine-containing DNA substrates [Nonekowski02]. Structural requirements of the preQ1 substrate and implications with respect to the catalytic mechanism are discussed [Hoops95].

A tgt mutant shows a defect in queuosine modification of tRNA and somewhat more rapid growth and higher sensitivity to suboptimal conditions [Noguchi82]. A mutant shows decreased recognition of a UAG translational stop [Frey89] and a defect in maintenance of the proper reading frame during translation [Urbonavicius01]. Under anaerobic conditions, a tgt mutant is defective in nitrate respiration [Janel84].

Reviews: [IwataReuyl03, Stengl05]

Citations: [Chong94, Dineshkumar02, Urbonavicius03]

Gene Citations: [Pogliano94a]

Locations: cytosol

Map Position: [425,361 -> 426,488] (9.17 centisomes)
Length: 1128 bp / 375 aa

Molecular Weight of Polypeptide: 42.594 kD (from nucleotide sequence), 46.0 kD (experimental) [Reuter91 ]

Unification Links: ASAP:ABE-0001413 , CGSC:118 , EchoBASE:EB0989 , EcoGene:EG10996 , OU-Microarray:b0406 , PortEco:tgt , RegulonDB:EG10996

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0002099 - tRNA wobble guanine modification Inferred from experiment [Noguchi82]
GO:0008616 - queuosine biosynthetic process Inferred from experiment [Noguchi82]
Molecular Function: GO:0008270 - zinc ion binding Inferred from experiment [Chong95]
GO:0008479 - queuine tRNA-ribosyltransferase activity Inferred from experiment [Garcia93a]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08]
GO:0005737 - cytoplasm

MultiFun Terms: information transfer RNA related RNA modification

Essentiality data for tgt 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]

Credits:
Last-Curated ? 08-Sep-2011 by Keseler I , SRI International


Enzymatic reaction of: tRNA-guanine transglycosylase

EC Number: 2.4.2.29

a guanine34 in tRNA + preQ1 <=> a 7-aminomethyl-7-deazaguanosine34 in tRNA + guanine

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.

In Pathways: queuosine biosynthesis

Summary:
The enzyme was first purified from E. coli B [Okada79].

Activity is sensitive to crosslinking reagents [Garcia93a] and thiol reagents [Garcia97].

Cofactors or Prosthetic Groups: Zn2+ [Chong95]

Inhibitors (Competitive): 7-methyl-preQ0 [Hoops95] , FMPP [Hoops95a]

Inhibitors (Unknown Mechanism): dimethylsuberimidate [Garcia93a] , N-ethylmaleimide [Garcia97] , methylmethanethiosulfonate [Garcia97] , 5,5'-dithio-bis-2-nitrobenzoate [Garcia97] , ethylacetimidate [Garcia93a]

Kinetic Parameters:

Substrate
Km (μM)
Citations
guanine
0.099
[GoodenoughLashu03]
a guanine34 in tRNA
0.12
[GoodenoughLashu03]


Sequence Features

Feature Class Location Citations Comment
Mutagenesis-Variant 28
[UniProt10]
Alternate sequence: C → A; UniProt: Slight loss of activity;
Sequence-Conflict 54 -> 55
[Garcia93a, Reuter91, UniProt10]
Alternate sequence: IL → MV; UniProt: (in Ref. 1 and 2);
Active-Site 89
[UniProt10]
UniProt: Nucleophile;
Mutagenesis-Variant 90
[UniProt10]
Alternate sequence: S → F; UniProt: Loss of activity;
Alternate sequence: S → C; UniProt: Loss of activity;
Alternate sequence: S → A; UniProt: Loss of activity;
Amino-Acid-Sites-That-Bind 90
[UniProt10]
UniProt: Substrate;
Mutagenesis-Variant 145
[UniProt10]
Alternate sequence: C → A; UniProt: No loss of activity;
Mutagenesis-Variant 232
[UniProt10]
Alternate sequence: C → A; UniProt: Slight loss of activity;
Mutagenesis-Variant 265
[UniProt10]
Alternate sequence: C → A; UniProt: Significant loss of activity;
Mutagenesis-Variant 302
[UniProt10]
Alternate sequence: C → A; UniProt: Loss of activity;
Metal-Binding-Site 302
[UniProt10]
UniProt: Zinc;
Mutagenesis-Variant 304
[UniProt10]
Alternate sequence: C → A; UniProt: Loss of activity;
Metal-Binding-Site 304
[UniProt10]
UniProt: Zinc;
Mutagenesis-Variant 307
[UniProt10]
Alternate sequence: C → A; UniProt: Loss of activity;
Metal-Binding-Site 307
[UniProt10]
UniProt: Zinc;
Mutagenesis-Variant 316
[UniProt10]
Alternate sequence: H → A; UniProt: Slight loss of activity;
Mutagenesis-Variant 317
[UniProt10]
Alternate sequence: H → A; UniProt: Loss of activity;
Mutagenesis-Variant 321
[UniProt10]
Alternate sequence: C → A; UniProt: No loss of activity;
Metal-Binding-Site 333
[UniProt10a]
UniProt: Zinc; Non-Experimental Qualifier: by similarity;


Gene Local Context (not to scale): ?

Transcription Units:

Notes:

History:
10/20/97 Gene b0406 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10996; confirmed by SwissProt match.


References

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

Chen11d: Chen YC, Brooks AF, Goodenough-Lashua DM, Kittendorf JD, Showalter HD, Garcia GA (2011). "Evolution of eukaryal tRNA-guanine transglycosylase: insight gained from the heterocyclic substrate recognition by the wild-type and mutant human and Escherichia coli tRNA-guanine transglycosylases." Nucleic Acids Res 39(7);2834-44. PMID: 21131277

Chong94: Chong S, Garcia GA (1994). "A versatile and general prokaryotic expression vector, pLACT7." Biotechniques 17(4);686, 688, 690-1. PMID: 7833029

Chong95: Chong S, Curnow AW, Huston TJ, Garcia GA (1995). "tRNA-guanine transglycosylase from Escherichia coli is a zinc metalloprotein. Site-directed mutagenesis studies to identify the zinc ligands." Biochemistry 34(11);3694-701. PMID: 7893665

Curnow93: Curnow AW, Kung FL, Koch KA, Garcia GA (1993). "tRNA-guanine transglycosylase from Escherichia coli: gross tRNA structural requirements for recognition." Biochemistry 32(19);5239-46. PMID: 8494901

Curnow94: Curnow AW, Garcia GA (1994). "tRNA-guanine transglycosylase from Escherichia coli: recognition of dimeric, unmodified tRNA(Tyr)." Biochimie 76(12);1183-91. PMID: 7748954

Curnow95: Curnow AW, Garcia GA (1995). "tRNA-guanine transglycosylase from Escherichia coli. Minimal tRNA structure and sequence requirements for recognition." J Biol Chem 270(29);17264-7. PMID: 7615526

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

Dineshkumar02: Dineshkumar TK, Thanedar S, Subbulakshmi C, Varshney U (2002). "An unexpected absence of queuosine modification in the tRNAs of an Escherichia coli B strain." Microbiology 148(Pt 12);3779-87. PMID: 12480882

Eric11: Eric Thomas C, Chen YC, Garcia GA (2011). "Differential heterocyclic substrate recognition by, and pteridine inhibition of E. coli and human tRNA-guanine transglycosylases." Biochem Biophys Res Commun 410(1);34-9. PMID: 21640076

Frey89: Frey B, Janel G, Michelsen U, Kersten H (1989). "Mutations in the Escherichia coli fnr and tgt genes: control of molybdate reductase activity and the cytochrome d complex by fnr." J Bacteriol 171(3);1524-30. PMID: 2537821

Garcia09: Garcia GA, Chervin SM, Kittendorf JD (2009). "Identification of the rate-determining step of tRNA-guanine transglycosylase from Escherichia coli." Biochemistry 48(47);11243-51. PMID: 19874048

Garcia93a: Garcia GA, Koch KA, Chong S (1993). "tRNA-guanine transglycosylase from Escherichia coli. Overexpression, purification and quaternary structure." J Mol Biol 231(2);489-97. PMID: 8323579

Garcia96a: Garcia GA, Tierney DL, Chong S, Clark K, Penner-Hahn JE (1996). "X-ray absorption spectroscopy of the zinc site in tRNA-guanine transglycosylase from Escherichia coli." Biochemistry 35(9);3133-9. PMID: 8608154

Garcia97: Garcia GA, Chong S (1997). "Cysteine 265 is in the active site of, but is not essential for catalysis by tRNA-guanine transglycosylase (TGT) from Escherichia coli." J Protein Chem 16(1);11-7. PMID: 9055203

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

GoodenoughLashu03: Goodenough-Lashua DM, Garcia GA (2003). "tRNA-guanine transglycosylase from E. coli: a ping-pong kinetic mechanism is consistent with nucleophilic catalysis." Bioorg Chem 31(4);331-44. PMID: 12877882

Hoops95: Hoops GC, Townsend LB, Garcia GA (1995). "tRNA-guanine transglycosylase from Escherichia coli: structure-activity studies investigating the role of the aminomethyl substituent of the heterocyclic substrate PreQ1." Biochemistry 34(46);15381-7. PMID: 7578154

Hoops95a: Hoops GC, Townsend LB, Garcia GA (1995). "Mechanism-based inactivation of tRNA-guanine transglycosylase from Escherichia coli by 2-amino-5-(fluoromethyl)pyrrolo[2,3-d]pyrimidin-4 (3H)-one." Biochemistry 34(47);15539-44. PMID: 7492556

Ishihama08: Ishihama Y, Schmidt T, Rappsilber J, Mann M, Hartl FU, Kerner MJ, Frishman D (2008). "Protein abundance profiling of the Escherichia coli cytosol." BMC Genomics 9;102. PMID: 18304323

IwataReuyl03: Iwata-Reuyl D (2003). "Biosynthesis of the 7-deazaguanosine hypermodified nucleosides of transfer RNA." Bioorg Chem 31(1);24-43. PMID: 12697167

Janel84: Janel G, Michelsen U, Nishimura S, Kersten H (1984). "Queuosine modification in tRNA and expression of the nitrate reductase in Escherichia coli." EMBO J 3(7);1603-8. PMID: 6204866

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

Kittendorf01: Kittendorf JD, Barcomb LM, Nonekowski ST, Garcia GA (2001). "tRNA-guanine transglycosylase from Escherichia coli: molecular mechanism and role of aspartate 89." Biochemistry 40(47);14123-33. PMID: 11714265

Kittendorf03: Kittendorf JD, Sgraja T, Reuter K, Klebe G, Garcia GA (2003). "An essential role for aspartate 264 in catalysis by tRNA-guanine transglycosylase from Escherichia coli." J Biol Chem 278(43);42369-76. PMID: 12909636

Kung00: Kung FL, Nonekowski S, Garcia GA (2000). "tRNA-guanine transglycosylase from Escherichia coli: recognition of noncognate-cognate chimeric tRNA and discovery of a novel recognition site within the TpsiC arm of tRNA(Phe)." RNA 6(2);233-44. PMID: 10688362

Kung98: Kung FL, Garcia GA (1998). "tRNA-guanine transglycosylase from Escherichia coli: recognition of full-length 'queuine-cognate' tRNAs." FEBS Lett 431(3);427-32. PMID: 9714557

Mueller95: Mueller SO, Slany RK (1995). "Structural analysis of the interaction of the tRNA modifying enzymes Tgt and QueA with a substrate tRNA." FEBS Lett 361(2-3);259-64. PMID: 7698334

Nakanishi94: Nakanishi S, Ueda T, Hori H, Yamazaki N, Okada N, Watanabe K (1994). "A UGU sequence in the anticodon loop is a minimum requirement for recognition by Escherichia coli tRNA-guanine transglycosylase." J Biol Chem 269(51);32221-5. PMID: 7528209

Noguchi82: Noguchi S, Nishimura Y, Hirota Y, Nishimura S (1982). "Isolation and characterization of an Escherichia coli mutant lacking tRNA-guanine transglycosylase. Function and biosynthesis of queuosine in tRNA." J Biol Chem 257(11);6544-50. PMID: 6804468

Nonekowski01: Nonekowski ST, Garcia GA (2001). "tRNA recognition by tRNA-guanine transglycosylase from Escherichia coli: the role of U33 in U-G-U sequence recognition." RNA 7(10);1432-41. PMID: 11680848

Nonekowski02: Nonekowski ST, Kung FL, Garcia GA (2002). "The Escherichia coli tRNA-guanine transglycosylase can recognize and modify DNA." J Biol Chem 277(9);7178-82. PMID: 11751936

Okada79: Okada N, Nishimura S (1979). "Isolation and characterization of a guanine insertion enzyme, a specific tRNA transglycosylase, from Escherichia coli." J Biol Chem 254(8);3061-6. PMID: 107167

Pogliano94a: Pogliano KJ, Beckwith J (1994). "Genetic and molecular characterization of the Escherichia coli secD operon and its products." J Bacteriol 176(3);804-14. PMID: 7507921

Reuter91: Reuter K, Slany R, Ullrich F, Kersten H (1991). "Structure and organization of Escherichia coli genes involved in biosynthesis of the deazaguanine derivative queuine, a nutrient factor for eukaryotes." J Bacteriol 173(7);2256-64. PMID: 1706703

Reuter94: Reuter K, Chong S, Ullrich F, Kersten H, Garcia GA (1994). "Serine 90 is required for enzymic activity by tRNA-guanine transglycosylase from Escherichia coli." Biochemistry 33(23);7041-6. PMID: 8003468

Reuter95: Reuter K, Ficner R (1995). "Sequence analysis and overexpression of the Zymomonas mobilis tgt gene encoding tRNA-guanine transglycosylase: purification and biochemical characterization of the enzyme." J Bacteriol 177(18);5284-8. PMID: 7665516

Stengl05: Stengl B, Reuter K, Klebe G (2005). "Mechanism and substrate specificity of tRNA-guanine transglycosylases (TGTs): tRNA-modifying enzymes from the three different kingdoms of life share a common catalytic mechanism." Chembiochem 6(11);1926-39. PMID: 16206323

Todorov05: Todorov KA, Tan XJ, Nonekowski ST, Garcia GA, Carlson HA (2005). "The role of aspartic acid 143 in E. coli tRNA-guanine transglycosylase: insights from mutagenesis studies and computational modeling." Biophys J 89(3);1965-77. PMID: 15951383

Todorov06: Todorov KA, Garcia GA (2006). "Role of aspartate 143 in Escherichia coli tRNA-guanine transglycosylase: alteration of heterocyclic substrate specificity." Biochemistry 45(2);617-25. PMID: 16401090

UniProt10: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

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

Urbonavicius01: Urbonavicius J, Qian Q, Durand JM, Hagervall TG, Bjork GR (2001). "Improvement of reading frame maintenance is a common function for several tRNA modifications." EMBO J 20(17);4863-73. PMID: 11532950

Urbonavicius03: Urbonavicius J, Stahl G, Durand JM, Ben Salem SN, Qian Q, Farabaugh PJ, Bjork GR (2003). "Transfer RNA modifications that alter +1 frameshifting in general fail to affect -1 frameshifting." RNA 9(6);760-8. PMID: 12756333

Other References Related to Gene Regulation

Salmon03: Salmon K, Hung SP, Mekjian K, Baldi P, Hatfield GW, Gunsalus RP (2003). "Global gene expression profiling in Escherichia coli K12. The effects of oxygen availability and FNR." J Biol Chem 278(32);29837-55. PMID: 12754220


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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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