1KCE

E. COLI THYMIDYLATE SYNTHASE MUTANT E58Q IN COMPLEX WITH CB3717 AND 2'-DEOXYURIDINE 5'-MONOPHOSPHATE (DUMP)


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.00 Å
  • R-Value Free: 0.222 
  • R-Value Work: 0.172 
  • R-Value Observed: 0.172 

wwPDB Validation   3D Report Full Report


This is version 1.2 of the entry. See complete history


Literature

An essential role for water in an enzyme reaction mechanism: the crystal structure of the thymidylate synthase mutant E58Q.

Sage, C.R.Rutenber, E.E.Stout, T.J.Stroud, R.M.

(1996) Biochemistry 35: 16270-16281

  • DOI: https://doi.org/10.1021/bi961269r
  • Primary Citation of Related Structures:  
    1KCE, 1ZPR

  • PubMed Abstract: 

    A water-mediated hydrogen bond network coordinated by glutamate 60(58) appears to play an important role in the thymidylate synthase (TS) reaction mechanism. We have addressed the role of glutamate 60(58) in the TS reaction by cocrystalizing the Escherichia coli TS mutant E60(58)Q with dUMP and the cofactor analog CB3717 and have determined the X-ray crystal structure to 2.5 A resolution with a final R factor of 15.2% (Rfree = 24.0%). Using difference Fourier analysis, we analyzed directly the changes that occur between wild-type and mutant structures. The structure of the mutant enzyme suggests that E60(58) is not required to properly position the ligands in the active site and that the coordinated hydrogen bond network has been disrupted in the mutant, providing an atomic resolution explanation for the impairment of the TS reaction by the E60(58)Q mutant and confirming the proposal that E60(58) coordinates this conserved hydrogen bond network. The structure also provides insight into the role of specific waters in the active site which have been suggested to be important in the TS reaction. Finally, the structure shows a unique conformation for the cofactor analog, CB3717, which has implications for structure-based drug design and sheds light on the controversy surrounding the previously observed enzymatic nonidentity between the chemically identical monomers of the TS dimer.


  • Organizational Affiliation

    Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
THYMIDYLATE SYNTHASE
A, B
264Escherichia coliMutation(s): 0 
Gene Names: THYA
EC: 2.1.1.45
UniProt
Find proteins for P0A884 (Escherichia coli (strain K12))
Explore P0A884 
Go to UniProtKB:  P0A884
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP0A884
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 2 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
CB3
Query on CB3

Download Ideal Coordinates CCD File 
D [auth A],
F [auth B]
10-PROPARGYL-5,8-DIDEAZAFOLIC ACID
C24 H23 N5 O6
LTKHPMDRMUCUEB-IBGZPJMESA-N
UMP
Query on UMP

Download Ideal Coordinates CCD File 
C [auth A],
E [auth B]
2'-DEOXYURIDINE 5'-MONOPHOSPHATE
C9 H13 N2 O8 P
JSRLJPSBLDHEIO-SHYZEUOFSA-N
Modified Residues  1 Unique
IDChains TypeFormula2D DiagramParent
CXM
Query on CXM
A, B
L-PEPTIDE LINKINGC6 H11 N O4 SMET
Binding Affinity Annotations 
IDSourceBinding Affinity
CB3 BindingDB:  1KCE IC50: 60 (nM) from 1 assay(s)
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.00 Å
  • R-Value Free: 0.222 
  • R-Value Work: 0.172 
  • R-Value Observed: 0.172 
  • Space Group: P 63
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 126.8α = 90
b = 126.8β = 90
c = 67.6γ = 120
Software Package:
Software NamePurpose
X-PLORmodel building
X-PLORrefinement
DENZOdata reduction
X-PLORphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 1997-04-21
    Type: Initial release
  • Version 1.1: 2008-03-24
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Non-polymer description, Version format compliance