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PDBsum entry 1wkf

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protein metals links
tRNA-modifying enzyme PDB id
1wkf
Jmol
Contents
Protein chain
372 a.a. *
Metals
_ZN
Waters ×279
* Residue conservation analysis
PDB id:
1wkf
Name: tRNA-modifying enzyme
Title: tRNA-guanine transglycosylase
Structure: tRNA-guanine transglycosylase. Chain: a. Synonym: tgt. Engineered: yes. Mutation: yes
Source: Zymomonas mobilis. Organism_taxid: 542. Gene: tgt. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Dimer (from PQS)
Resolution:
2.20Å     R-factor:   0.199     R-free:   0.250
Authors: C.Romier,K.Reuter,D.Suck,R.Ficner
Key ref:
C.Romier et al. (1996). Mutagenesis and crystallographic studies of Zymomonas mobilis tRNA-guanine transglycosylase reveal aspartate 102 as the active site nucleophile. Biochemistry, 35, 15734-15739. PubMed id: 8961936 DOI: 10.1021/bi962003n
Date:
06-Aug-96     Release date:   07-Jul-97    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
P28720  (TGT_ZYMMO) -  Queuine tRNA-ribosyltransferase
Seq:
Struc:
386 a.a.
372 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 2 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.2.4.2.29  - tRNA-guanine(34) transglycosylase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction:
1. Guanine34 in tRNA + queuine = queuosine34 in tRNA + guanine
2. Guanine34 in tRNA + 7-aminomethyl-7-carbaguanine = 7-aminomethyl-7- carbaguanine34 in tRNA + guanine
Guanine(34) in tRNA
+ queuine
= queuosine(34) in tRNA
+ guanine
Guanine(34) in tRNA
+ 7-aminomethyl-7-carbaguanine
= 7-aminomethyl-7- carbaguanine(34) in tRNA
+ guanine
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     tRNA processing   3 terms 
  Biochemical function     transferase activity     4 terms  

 

 
    reference    
 
 
DOI no: 10.1021/bi962003n Biochemistry 35:15734-15739 (1996)
PubMed id: 8961936  
 
 
Mutagenesis and crystallographic studies of Zymomonas mobilis tRNA-guanine transglycosylase reveal aspartate 102 as the active site nucleophile.
C.Romier, K.Reuter, D.Suck, R.Ficner.
 
  ABSTRACT  
 
Procaryotic tRNA-guanine transglycosylase (TGT) catalyzes the posttranscriptional base exchange of the queuine precursor 7-aminomethyl-7-deazaguanine (preQ1) with the genetically encoded guanine at the wobble position of tRNAs specific for Asn, Asp, His, and Tyr. The X-ray structures of Zymomonas mobilis TGT and of its complex with preQ1 [Romier, C., Reuter, K., Suck, D., & Ficner, R. (1996) EMBO J. 15, 2850-2857] have revealed a specific preQ1 binding pocket and allowed a proposal for tRNA binding and recognition. We have used band-shift experiments in denaturing conditions to study the enzymatic reaction performed by TGT. The presence of shifted protein bands after incubation with tRNA followed by protein denaturation indicates a reaction mechanism involving a covalent intermediate. Inspection of the X-ray structures and comparison of the different procaryotic TGT sequences highlighted the conserved aspartate 102 as the most likely nucleophile. Mutation of this residue into alanine by site-directed mutagenesis leads to an inactive mutant unable to form a covalent intermediate with tRNA, proving that aspartate 102 is the active site nucleophile in TGT. To investigate the recognition of the wobble guanine in the preQ1 binding pocket, we mutated aspartate 156, the major recognition element for preQ1, into alanine and tyrosine. Both mutants are inactive in producing the final product, but the mutant D156A is able to form the covalent intermediate with tRNA in the first step of the reaction mechanism in comparable amounts to wild-type protein. Therefore, the binding of the wobble guanine in the preQ1 binding pocket is required for the cleavage of the glycosidic bond. The three mutants were crystallized and their X-ray structures determined. The mutants display only subtle changes to the wild-type protein, confirming that the observed biochemical results are due to the chemical substitutions rather than structural rearrangements.
 

Literature references that cite this PDB file's key reference

  PubMed id Reference
19925456 M.Vinayak, and C.Pathak (2010).
Queuosine modification of tRNA: its divergent role in cellular machinery.
  Biosci Rep, 30, 135-148.  
19874048 G.A.Garcia, S.M.Chervin, and J.D.Kittendorf (2009).
Identification of the rate-determining step of tRNA-guanine transglycosylase from Escherichia coli.
  Biochemistry, 48, 11243-11251.  
19894214 T.Ritschel, P.C.Kohler, G.Neudert, A.Heine, F.Diederich, and G.Klebe (2009).
How to Replace the Residual Solvation Shell of Polar Active Site Residues to Achieve Nanomolar Inhibition of tRNA-Guanine Transglycosylase.
  ChemMedChem, 4, 2012-2023.
PDB codes: 3eos 3eou 3gc4 3gc5 3ge7
17673081 S.M.Chervin, J.D.Kittendorf, and G.A.Garcia (2007).
Probing the intermediacy of covalent RNA enzyme complexes in RNA modification enzymes.
  Methods Enzymol, 425, 121-137.  
16206323 B.Stengl, K.Reuter, and G.Klebe (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, 1926-1939.  
15888313 G.A.Garcia, and J.D.Kittendorf (2005).
Transglycosylation: a mechanism for RNA modification (and editing?).
  Bioorg Chem, 33, 229-251.  
15951383 K.A.Todorov, X.J.Tan, S.T.Nonekowski, G.A.Garcia, and H.A.Carlson (2005).
The role of aspartic acid 143 in E. coli tRNA-guanine transglycosylase: insights from mutagenesis studies and computational modeling.
  Biophys J, 89, 1965-1977.  
12581659 A.R.Ferré-D'Amaré (2003).
RNA-modifying enzymes.
  Curr Opin Struct Biol, 13, 49-55.  
14513020 C.C.Correll (2003).
Caught in the act of modifying tRNA.
  Nat Struct Biol, 10, 772-773.  
12877882 D.M.Goodenough-Lashua, and G.A.Garcia (2003).
tRNA-guanine transglycosylase from E. coli: a ping-pong kinetic mechanism is consistent with nucleophilic catalysis.
  Bioorg Chem, 31, 331-344.  
12909636 J.D.Kittendorf, T.Sgraja, K.Reuter, G.Klebe, and G.A.Garcia (2003).
An essential role for aspartate 264 in catalysis by tRNA-guanine transglycosylase from Escherichia coli.
  J Biol Chem, 278, 42369-42376.
PDB code: 1pxg
14523925 R.Brenk, M.T.Stubbs, A.Heine, K.Reuter, and G.Klebe (2003).
Flexible adaptations in the structure of the tRNA-modifying enzyme tRNA-guanine transglycosylase and their implications for substrate selectivity, reaction mechanism and structure-based drug design.
  Chembiochem, 4, 1066-1077.
PDB codes: 1ozm 1ozq 1p0b 1p0d 1p0e
12533518 S.G.Van Lanen, S.D.Kinzie, S.Matthieu, T.Link, J.Culp, and D.Iwata-Reuyl (2003).
tRNA modification by S-adenosylmethionine:tRNA ribosyltransferase-isomerase. Assay development and characterization of the recombinant enzyme.
  J Biol Chem, 278, 10491-10499.  
12949492 W.Xie, X.Liu, and R.H.Huang (2003).
Chemical trapping and crystal structure of a catalytic tRNA guanine transglycosylase covalent intermediate.
  Nat Struct Biol, 10, 781-788.
PDB codes: 1q2r 1q2s
11751936 S.T.Nonekowski, F.L.Kung, and G.A.Garcia (2002).
The Escherichia coli tRNA-guanine transglycosylase can recognize and modify DNA.
  J Biol Chem, 277, 7178-7182.  
  11680848 S.T.Nonekowski, and G.A.Garcia (2001).
tRNA recognition by tRNA-guanine transglycosylase from Escherichia coli: the role of U33 in U-G-U sequence recognition.
  RNA, 7, 1432-1441.  
10739928 C.Grimm, G.Klebe, R.Ficner, and K.Reuter (2000).
Screening orthologs as an important variable in crystallization: preliminary X-ray diffraction studies of the tRNA-modifying enzyme S-adenosyl-methionine:tRNA ribosyl transferase/isomerase.
  Acta Crystallogr D Biol Crystallogr, 56, 484-488.  
10688362 F.L.Kung, S.Nonekowski, and G.A.Garcia (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, 233-244.  
9572842 M.Degano, S.C.Almo, J.C.Sacchettini, and V.L.Schramm (1998).
Trypanosomal nucleoside hydrolase. A novel mechanism from the structure with a transition-state inhibitor.
  Biochemistry, 37, 6277-6285.
PDB code: 2mas
9667869 V.L.Schramm (1997).
Enzymatic N-riboside scission in RNA and RNA precursors.
  Curr Opin Chem Biol, 1, 323-331.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.