1JT9

Structure of the mutant F174A T form of the Glucosamine-6-Phosphate deaminase from E.coli


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.06 Å
  • R-Value Free: 0.224 
  • R-Value Work: 0.204 

wwPDB Validation   3D Report Full Report


This is version 1.5 of the entry. See complete history


Literature

On the role of the conformational flexibility of the active-site lid on the allosteric kinetics of glucosamine-6-phosphate deaminase.

Bustos-Jaimes, I.Sosa-Peinado, A.Rudino-Pinera, E.Horjales, E.Calcagno, M.L.

(2002) J Mol Biol 319: 183-189

  • DOI: https://doi.org/10.1016/S0022-2836(02)00096-7
  • Primary Citation of Related Structures:  
    1JT9

  • PubMed Abstract: 

    The active site of glucosamine-6-phosphate deaminase from Escherichia coli (GlcN6P deaminase, EC 3.5.99.6) has a complex lid formed by two antiparallel beta-strands connected by a helix-loop segment (158-187). This motif contains Arg172, which is a residue involved in binding the substrate in the active-site, and three residues that are part of the allosteric site, Arg158, Lys160 and Thr161. This dual binding role of the motif forming the lid suggests that it plays a key role in the functional coupling between active and allosteric sites. Previous crystallographic work showed that the temperature coefficients of the active-site lid are very large when the enzyme is in its T allosteric state. These coefficients decrease in the R state, thus suggesting that this motif changes its conformational flexibility as a consequence of the allosteric transition. In order to explore the possible connection between the conformational flexibility of the lid and the function of the deaminase, we constructed the site-directed mutant Phe174-Ala. Phe174 is located at the C-end of the lid helix and its side-chain establishes hydrophobic interactions with the remainder of the enzyme. The crystallographic structure of the T state of Phe174-Ala deaminase, determined at 2.02 A resolution, shows no density for the segment 162-181, which is part of the active-site lid (PDB 1JT9). This mutant form of the enzyme is essentially inactive in the absence of the allosteric activator, N-acetylglucosamine-6-P although it recovers its activity up to the wild-type level in the presence of this ligand. Spectrometric and binding studies show that inactivity is due to the inability of the active-site to bind ligands when the allosteric site is empty. These data indicate that the conformational flexibility of the active-site lid critically alters the binding properties of the active site, and that the occupation of the allosteric site restores the lid conformational flexibility to a functional state.


  • Organizational Affiliation

    Laboratorio de Fisicoquímica e Ingeniería de Proteínas, Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, P.O. Box 70-159, Ciudad Universitaria, Mexico City, D.F. 04510, Mexico. ismaelb@servidor.unam.mx


Macromolecules
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Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Glucosamine-6-Phosphate deaminase266Escherichia coliMutation(s): 1 
EC: 3.5.99.6
UniProt
Find proteins for P0A759 (Escherichia coli (strain K12))
Explore P0A759 
Go to UniProtKB:  P0A759
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP0A759
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.06 Å
  • R-Value Free: 0.224 
  • R-Value Work: 0.204 
  • Space Group: P 63 2 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 127.617α = 90
b = 127.617β = 90
c = 139.822γ = 120
Software Package:
Software NamePurpose
CNSrefinement
MAR345data collection
CCP4data scaling
CNSphasing

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2002-02-20
    Type: Initial release
  • Version 1.1: 2008-04-27
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Derived calculations, Version format compliance
  • Version 1.3: 2017-10-04
    Changes: Refinement description
  • Version 1.4: 2021-11-10
    Changes: Database references
  • Version 1.5: 2023-10-25
    Changes: Data collection, Refinement description