1PXG

Crystal structure of the mutated tRNA-guanine transglycosylase (TGT) D280E complexed with preQ1


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.70 Å
  • R-Value Free: 0.206 
  • R-Value Work: 0.159 
  • R-Value Observed: 0.159 

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Ligand Structure Quality Assessment 


This is version 1.6 of the entry. See complete history


Literature

An essential role for aspartate 264 in catalysis by tRNA-guanine transglycosylase from Escherichia coli.

Kittendorf, J.D.Sgraja, T.Reuter, K.Klebe, G.Garcia, G.A.

(2003) J Biol Chem 278: 42369-42376

  • DOI: https://doi.org/10.1074/jbc.M304323200
  • Primary Citation of Related Structures:  
    1PXG

  • PubMed Abstract: 

    tRNA-guanine transglycosylase (TGT) catalyzes a post-transcriptional base-exchange reaction involved in the incorporation of the modified base queuine (Q) into the wobble position of certain tRNAs. Catalysis by TGT occurs through a double-displacement mechanism that involves the formation of a covalent enzyme-RNA intermediate (Kittendorf, J. D., Barcomb, L. M., Nonekowski, S. T., and Garcia, G. A. (2001) Biochemistry 40, 14123-14133). The TGT chemical mechanism requires the protonation of the displaced guanine and the deprotonation of the incoming heterocyclic base. Based on its position in the active site, it is likely that aspartate 264 is involved in these proton transfer events. To investigate this possibility, site-directed mutagenesis was employed to convert aspartate 264 to alanine, asparagine, glutamate, glutamine, lysine, and histidine. Biochemical characterization of these TGT mutants revealed that only the conservative glutamate mutant retained catalytic activity, with Km values for both tRNA and guanine 3-fold greater than those for wild-type, whereas the kcat was depressed by an order of magnitude. Furthermore, of these six TGT mutants, only the TGT(D264E) was capable of forming a TGT.RNA covalent intermediate; however, unlike wild-type TGT, only hydroxylamine is capable of cleaving the TGT(D264E).RNA covalent complex. In an effort to better understand the unique biochemical properties of the D264E TGT mutant, we solved the crystal structure of the Zymomonas mobilis TGT with the analogous mutation (D280E). The results of these studies support two roles for aspartate 264 in catalysis by TGT, protonation of the leaving guanine and deprotonation of the incoming preQ1.


  • Organizational Affiliation

    Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, 428 Chuirch Street, Ann Arbor, MI 48109-1065, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Queuine tRNA-ribosyltransferase382Zymomonas mobilisMutation(s): 1 
Gene Names: TGT
EC: 2.4.2.29
UniProt
Find proteins for P28720 (Zymomonas mobilis subsp. mobilis (strain ATCC 31821 / ZM4 / CP4))
Explore P28720 
Go to UniProtKB:  P28720
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP28720
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 3 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
PRF
Query on PRF

Download Ideal Coordinates CCD File 
C [auth A]7-DEAZA-7-AMINOMETHYL-GUANINE
C7 H9 N5 O
MEYMBLGOKYDGLZ-UHFFFAOYSA-N
GOL
Query on GOL

Download Ideal Coordinates CCD File 
D [auth A]
E [auth A]
F [auth A]
G [auth A]
H [auth A]
D [auth A],
E [auth A],
F [auth A],
G [auth A],
H [auth A],
I [auth A],
J [auth A],
K [auth A]
GLYCEROL
C3 H8 O3
PEDCQBHIVMGVHV-UHFFFAOYSA-N
ZN
Query on ZN

Download Ideal Coordinates CCD File 
B [auth A]ZINC ION
Zn
PTFCDOFLOPIGGS-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.70 Å
  • R-Value Free: 0.206 
  • R-Value Work: 0.159 
  • R-Value Observed: 0.159 
  • Space Group: C 1 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 91.28α = 90
b = 64.94β = 96.12
c = 70.2γ = 90
Software Package:
Software NamePurpose
SHELXmodel building
SHELXL-97refinement
SCALEPACKdata scaling
SHELXphasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2003-09-09
    Type: Initial release
  • Version 1.1: 2008-04-29
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Derived calculations, Version format compliance
  • Version 1.3: 2017-10-11
    Changes: Refinement description
  • Version 1.4: 2020-08-19
    Changes: Advisory, Derived calculations
  • Version 1.5: 2021-10-27
    Changes: Database references
  • Version 1.6: 2023-08-16
    Changes: Data collection, Refinement description