3IX9

Crystal structure of Streptococcus pneumoniae dihydrofolate reductase - Sp9 mutant


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.95 Å
  • R-Value Free: 0.274 
  • R-Value Work: 0.237 
  • R-Value Observed: 0.237 

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This is version 1.3 of the entry. See complete history


Literature

Kinetic and structural characterization of dihydrofolate reductase from Streptococcus pneumoniae

Lee, J.Yennawar, N.H.Gam, J.Benkovic, S.J.

(2010) Biochemistry 49: 195-206

  • DOI: https://doi.org/10.1021/bi901614m
  • Primary Citation of Related Structures:  
    3IX9

  • PubMed Abstract: 

    Drug resistance associated with dihydrofolate reductase (DHFR) has emerged as a critical issue in the treatment of bacterial infections. In our efforts to understand the mechanism of a drug-resistant dihydrofolate reductase (DHFR) from a pathogenic bacterial source, we report the first kinetic characterization of Streptococcus pneumoniae DHFR (spDHFR) along with its X-ray structure. This study revealed that the kinetic properties of spDHFR were significantly different from those of Escherichia coli DHFR. The product (tetrahydrofolate) dissociation step that is the rate-limiting step in E. coli DHFR is significantly accelerated in spDHFR so that hydride transfer or a preceding step is rate-limiting. Comparison of the binding parameters of this enzyme to those of a mutant spDHFR (Sp9) confirmed that the Leu100 residue in spDHFR is the critical element for the trimethoprim (TMP) resistance. Steady-state kinetics exhibited a pH dependence in k(cat), which prompted us to elucidate the role of the new catalytic residue (His33) in the active site of spDHFR. Structural data of the Sp9 mutant in complex with NADPH and methotrexate confirmed the participation of His33 in a hydrogen bonding network involving a water molecule, the hydroxyl group of Thr119, and the carboxylate ion of Glu30. Sequence analysis of the DHFR superfamily revealed that the His residue is the major amino acid component at this position and is found mostly in pathogenic bacterial DHFRs. A mutation of Val100 to Leu demonstrated a steric clash of the leucine side chain with the side chains of Ile8 and Phe34, rationalizing weaker binding of trimethoprim to Leu100 DHFR. Understanding the role of specific amino acids in the active site coupled with detailed structural analysis will inform us on how to better design inhibitors targeting drug-resistant pathogenic bacterial DHFRs.


  • Organizational Affiliation

    Department of Chemistry, 414 Wartik Laboratory, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Dihydrofolate reductase
A, B
190Streptococcus pneumoniaeMutation(s): 9 
Gene Names: dfr
EC: 1.5.1.3
UniProt
Find proteins for Q54801 (Streptococcus pneumoniae serotype 4 (strain ATCC BAA-334 / TIGR4))
Explore Q54801 
Go to UniProtKB:  Q54801
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ54801
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Binding Affinity Annotations 
IDSourceBinding Affinity
MTX Binding MOAD:  3IX9 Ki: 3.9 (nM) from 1 assay(s)
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.95 Å
  • R-Value Free: 0.274 
  • R-Value Work: 0.237 
  • R-Value Observed: 0.237 
  • Space Group: P 21 21 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 61.838α = 90
b = 93.639β = 90
c = 71.367γ = 90
Software Package:
Software NamePurpose
CrystalCleardata collection
PHASERphasing
CNSrefinement
CrystalCleardata reduction
CrystalCleardata scaling

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2010-02-02
    Type: Initial release
  • Version 1.1: 2011-07-13
    Changes: Version format compliance
  • Version 1.2: 2021-10-13
    Changes: Database references, Derived calculations
  • Version 1.3: 2023-09-06
    Changes: Data collection, Refinement description