1JMH

CONTRIBUTIONS OF ORIENTATION AND HYDROGEN BONDING TO CATALYSIS IN ASN-229 MUTANTS OF THYMIDYLATE SYNTHASE


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.50 Å
  • R-Value Free: 0.311 
  • R-Value Work: 0.213 
  • R-Value Observed: 0.213 

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Ligand Structure Quality Assessment 


This is version 1.5 of the entry. See complete history


Literature

Contributions of orientation and hydrogen bonding to catalysis in Asn229 mutants of thymidylate synthase.

Finer-Moore, J.S.Liu, L.Birdsall, D.L.Brem, R.Apfeld, J.Santi, D.V.Stroud, R.M.

(1998) J Mol Biol 276: 113-129

  • DOI: https://doi.org/10.1006/jmbi.1997.1495
  • Primary Citation of Related Structures:  
    1JMF, 1JMG, 1JMH, 1JMI, 1TVU, 1TVV, 1TVW

  • PubMed Abstract: 

    We have determined structures of binary and ternary complexes of five Asn229 variants of thymidylate synthase (TS) and related their structures to the kinetic constants measured previously. Asn229 forms two hydrogen bonds to the pyrimidine ring of the substrate 2'-deoxyuridine-5'-monophosphate (dUMP). These hydrogen bonds constrain the orientation of dUMP in binary complexes with dUMP, and in ternary complexes with dUMP and the TS cofactor, 5,10-methylene-5,6,7,8-tetrahydrofolate. In N229 mutants, where these hydrogen bonds cannot be made, dUMP binds in a misoriented or more disordered fashion. Most N229 mutants exhibit no activity for the dehalogenation of 5-bromo-dUMP, which requires correct orientation of dUMP against Cys198. Since bound dUMP forms the binding surface against which the pterin ring of cofactor binds, misorientation of dUMP results in higher Km values for cofactor. At the same time, binding of the cofactor aids in ordering and positioning dUMP for catalysis. Hydrophobic mutants, such as N229I, favor an arrangement of solvent molecules and side-chains around the ligands similar to that in a proposed transition state for ternary complex formation in wild-type TS, and kcat values are similar to the wild-type value. Smaller, more hydrophilic mutants favor arrangements of the solvent and side-chains surrounding the ligands that do not resemble the proposed transition state. These changes correspond to decreases in kcat of up to 2000-fold, with only modest increases in Km or Kd. These results are consistent with the proposal that the hydrogen-bonding network between water, dUMP and side-chains in the active-site cavity contributes to catalysis in TS. Asn229 has the unique ability to maintain this critical network, without sterically interfering with dUMP binding.


  • 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 SYNTHASE316Lacticaseibacillus caseiMutation(s): 1 
Gene Names: N229I MUTANT OF CLONED L. CASE
EC: 2.1.1.45
UniProt
Find proteins for P00469 (Lacticaseibacillus casei)
Explore P00469 
Go to UniProtKB:  P00469
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP00469
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 1 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
UMP
Query on UMP

Download Ideal Coordinates CCD File 
B [auth A]2'-DEOXYURIDINE 5'-MONOPHOSPHATE
C9 H13 N2 O8 P
JSRLJPSBLDHEIO-SHYZEUOFSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.50 Å
  • R-Value Free: 0.311 
  • R-Value Work: 0.213 
  • R-Value Observed: 0.213 
  • Space Group: P 61 2 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 78.4α = 90
b = 78.4β = 90
c = 242.5γ = 120
Software Package:
Software NamePurpose
X-PLORmodel building
X-PLORrefinement
R-AXISdata reduction
X-PLORphasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 1998-01-28
    Type: Initial release
  • Version 1.1: 2008-03-24
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Derived calculations, Version format compliance
  • Version 1.3: 2011-11-16
    Changes: Atomic model
  • Version 1.4: 2021-11-03
    Changes: Database references, Derived calculations, Other
  • Version 1.5: 2024-02-07
    Changes: Data collection