1ISO

ISOCITRATE DEHYDROGENASE: STRUCTURE OF AN ENGINEERED NADP+--> NAD+ SPECIFICITY-REVERSAL MUTANT


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.90 Å
  • R-Value Free: 0.218 
  • R-Value Work: 0.186 
  • R-Value Observed: 0.186 

wwPDB Validation   3D Report Full Report


This is version 1.4 of the entry. See complete history


Literature

Determinants of cofactor specificity in isocitrate dehydrogenase: structure of an engineered NADP+ --> NAD+ specificity-reversal mutant.

Hurley, J.H.Chen, R.Dean, A.M.

(1996) Biochemistry 35: 5670-5678

  • DOI: https://doi.org/10.1021/bi953001q
  • Primary Citation of Related Structures:  
    1ISO

  • PubMed Abstract: 

    The 7-fold mutation Cys201Met/Cys332Tyr/Lys344Asp/Tyr345Ile/Val35 1Ala/Tyr391Lys/Arg395Ser converts the cofactor specificity of Escherichia coli isocitrate dehydrogenase from a 7000-fold preference for NADP+ to a 200-fold preference for NAD+, with overall activity comparable to that of wild-type NAD+-dependent isocitrate dehydrogenases. The structure of the NAD+-dependent mutant has been determined and refined to a working R-factor of 0.186 at 1.9 A resolution. The structure shows that NADP+ affinity is destroyed by removing favorable interactions between the 2'-phosphate and Tyr345, Tyr391, and Arg395 and by adding a repulsive interaction with Asp344. NAD+ affinity is enhanced by adding hydrogen bonds between Asp344 and the free 2'-hydroxyl. The favorable Asp344-2'-OH interaction requires a change in the pucker of the ribose to C2' endo and a shift in the adenine ring. The ring shift is made possible by a series of changes in steric packing interactions. The linchpin for repacking in the adenosine binding site is residue 351. The side chain of this "second layer" residue dictates packing of the surrounding "first layer" residues which interact with the 2' moiety and, in turn, directly determine specificity.


  • Organizational Affiliation

    Laboratory of Molecular Biology, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0580, USA. HURLEY@TOVE.NIDDK.NIH.GOV


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
ISOCITRATE DEHYDROGENASE416Escherichia coliMutation(s): 7 
EC: 1.1.1.42
UniProt
Find proteins for P08200 (Escherichia coli (strain K12))
Explore P08200 
Go to UniProtKB:  P08200
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP08200
Sequence Annotations
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  • Reference Sequence
Small Molecules
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.90 Å
  • R-Value Free: 0.218 
  • R-Value Work: 0.186 
  • R-Value Observed: 0.186 
  • Space Group: P 43 21 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 102.4α = 90
b = 102.4β = 90
c = 150.6γ = 90
Software Package:
Software NamePurpose
X-PLORmodel building
X-PLORrefinement
DENZOdata reduction
SCALEPACKdata scaling
X-PLORphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

  • Released Date: 1996-12-07 
  • Deposition Author(s): Hurley, J.H.

Revision History  (Full details and data files)

  • Version 1.0: 1996-12-07
    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: 2021-11-03
    Changes: Database references, Derived calculations, Other
  • Version 1.4: 2023-08-09
    Changes: Refinement description