PDBsum entry 1iso

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protein ligands links
Oxidoreductase PDB id
Protein chain
414 a.a. *
SO4 ×3
Waters ×378
* Residue conservation analysis
PDB id:
Name: Oxidoreductase
Title: Isocitrate dehydrogenase: structure of an engineered NADP+-- specificity-reversal mutant
Structure: Isocitrate dehydrogenase. Chain: a. Synonym: oxalosuccinate decarboxylase, idh. Engineered: yes. Mutation: yes
Source: Escherichia coli. Organism_taxid: 562
Biol. unit: Dimer (from PQS)
1.90Å     R-factor:   0.186     R-free:   0.218
Authors: J.H.Hurley
Key ref:
J.H.Hurley et al. (1996). Determinants of cofactor specificity in isocitrate dehydrogenase: structure of an engineered NADP+ --> NAD+ specificity-reversal mutant. Biochemistry, 35, 5670-5678. PubMed id: 8639526 DOI: 10.1021/bi953001q
01-Mar-96     Release date:   07-Dec-96    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P08200  (IDH_ECOLI) -  Isocitrate dehydrogenase [NADP]
416 a.a.
414 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 7 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.  - Isocitrate dehydrogenase (NADP(+)).
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]

Citric acid cycle
      Reaction: Isocitrate + NADP+ = 2-oxoglutarate + CO2 + NADPH
Bound ligand (Het Group name = NAD)
matches with 47.92% similarity
= 2-oxoglutarate
+ CO(2)
      Cofactor: Mn(2+) or Mg(2+)
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     cytoplasm   1 term 
  Biological process     oxidation-reduction process   5 terms 
  Biochemical function     oxidoreductase activity     6 terms  


DOI no: 10.1021/bi953001q Biochemistry 35:5670-5678 (1996)
PubMed id: 8639526  
Determinants of cofactor specificity in isocitrate dehydrogenase: structure of an engineered NADP+ --> NAD+ specificity-reversal mutant.
J.H.Hurley, R.Chen, A.M.Dean.
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.

Literature references that cite this PDB file's key reference

  PubMed id Reference
19507198 M.Ehsani, M.R.Fernández, J.A.Biosca, and S.Dequin (2009).
Reversal of coenzyme specificity of 2,3-butanediol dehydrogenase from Saccharomyces cerevisae and in vivo functional analysis.
  Biotechnol Bioeng, 104, 381-389.  
19181802 T.Fuhrer, and U.Sauer (2009).
Different biochemical mechanisms ensure network-wide balancing of reducing equivalents in microbial metabolism.
  J Bacteriol, 191, 2112-2121.  
17634983 K.Imada, T.Tamura, R.Takenaka, I.Kobayashi, K.Namba, and K.Inagaki (2008).
Structure and quantum chemical analysis of NAD+-dependent isocitrate dehydrogenase: hydride transfer and co-factor specificity.
  Proteins, 70, 63-71.
PDB code: 2d4v
17251971 F.J.Poelwijk, D.J.Kiviet, D.M.Weinreich, and S.J.Tans (2007).
Empirical fitness landscapes reveal accessible evolutionary paths.
  Nature, 445, 383-386.  
16618920 J.M.Turner, J.Graziano, G.Spraggon, and P.G.Schultz (2006).
Structural plasticity of an aminoacyl-tRNA synthetase active site.
  Proc Natl Acad Sci U S A, 103, 6483-6488.
PDB codes: 1zh0 2ag6
16759231 M.Karlström, I.H.Steen, D.Madern, A.E.Fedöy, N.K.Birkeland, and R.Ladenstein (2006).
The crystal structure of a hyperthermostable subfamily II isocitrate dehydrogenase from Thermotoga maritima.
  FEBS J, 273, 2851-2868.
PDB code: 1zor
16767773 O.V.Kalinina, and M.S.Gelfand (2006).
Amino acid residues that determine functional specificity of NADP- and NAD-dependent isocitrate and isopropylmalate dehydrogenases.
  Proteins, 64, 1001-1009.  
16284723 A.Rodríguez-Arnedo, M.Camacho, F.Llorca, and M.J.Bonete (2005).
Complete reversal of coenzyme specificity of isocitrate dehydrogenase from Haloferax volcanii.
  Protein J, 24, 259-266.  
15653464 G.Zhu, G.B.Golding, and A.M.Dean (2005).
The selective cause of an ancient adaptation.
  Science, 307, 1279-1282.  
15623532 S.Watanabe, T.Kodaki, and K.Makino (2005).
Complete reversal of coenzyme specificity of xylitol dehydrogenase and increase of thermostability by the introduction of structural zinc.
  J Biol Chem, 280, 10340-10349.  
12562755 A.P.Lin, and L.McAlister-Henn (2003).
Homologous binding sites in yeast isocitrate dehydrogenase for cofactor (NAD+) and allosteric activator (AMP).
  J Biol Chem, 278, 12864-12872.  
14529270 R.Woodyer, W.A.van der Donk, and H.Zhao (2003).
Relaxing the nicotinamide cofactor specificity of phosphite dehydrogenase by rational design.
  Biochemistry, 42, 11604-11614.  
12855708 Y.Yasutake, S.Watanabe, M.Yao, Y.Takada, N.Fukunaga, and I.Tanaka (2003).
Crystal structure of the monomeric isocitrate dehydrogenase in the presence of NADP+: insight into the cofactor recognition, catalysis, and evolution.
  J Biol Chem, 278, 36897-36904.
PDB code: 1j1w
12204383 H.Inoue, T.Tamura, N.Ehara, A.Nishito, Y.Nakayama, M.Maekawa, K.Imada, H.Tanaka, and K.Inagaki (2002).
Biochemical and molecular characterization of the NAD(+)-dependent isocitrate dehydrogenase from the chemolithotroph Acidithiobacillus thiooxidans.
  FEMS Microbiol Lett, 214, 127-132.  
12454487 M.Karlström, I.H.Steen, G.Tibbelin, T.Lien, N.K.Birkeland, and R.Ladenstein (2002).
Crystallization and preliminary X-ray structure analysis of isocitrate dehydrogenase from two hyperthermophiles, Aeropyrum pernix and Thermotoga maritima.
  Acta Crystallogr D Biol Crystallogr, 58, 2162-2164.  
11969425 Y.C.Huang, and R.F.Colman (2002).
Evaluation by mutagenesis of the roles of His309, His315, and His319 in the coenzyme site of pig heart NADP-dependent isocitrate dehydrogenase.
  Biochemistry, 41, 5637-5643.  
11320304 C.E.Naylor, S.Gover, A.K.Basak, M.S.Cosgrove, H.R.Levy, and M.J.Adams (2001).
NADP+ and NAD+ binding to the dual coenzyme specific enzyme Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase: different interdomain hinge angles are seen in different binary and ternary complexes.
  Acta Crystallogr D Biol Crystallogr, 57, 635-648.
PDB codes: 1h93 1h94 1h9a 1h9b
11533060 I.H.Steen, D.Madern, M.Karlström, T.Lien, R.Ladenstein, and N.K.Birkeland (2001).
Comparison of isocitrate dehydrogenase from three hyperthermophiles reveals differences in thermostability, cofactor specificity, oligomeric state, and phylogenetic affiliation.
  J Biol Chem, 276, 43924-43931.  
11146097 R.Chen (2001).
Enzyme engineering: rational redesign versus directed evolution.
  Trends Biotechnol, 19, 13-14.  
11679744 Y.Yasutake, S.Watanabe, M.Yao, Y.Takada, N.Fukunaga, and I.Tanaka (2001).
Crystallization and preliminary X-ray diffraction studies of monomeric isocitrate dehydrogenase by the MAD method using Mn atoms.
  Acta Crystallogr D Biol Crystallogr, 57, 1682-1685.  
10869187 H.Wang, B.Lei, and S.C.Tu (2000).
Vibrio harveyi NADPH-FMN oxidoreductase arg203 as a critical residue for NADPH recognition and binding.
  Biochemistry, 39, 7813-7819.  
  11206056 R.Chen, and S.S.Jeong (2000).
Functional prediction: identification of protein orthologs and paralogs.
  Protein Sci, 9, 2344-2353.  
10801312 S.L.Anderson, K.I.Minard, and L.McAlister-Henn (2000).
Allosteric inhibition of NAD+-specific isocitrate dehydrogenase by a mitochondrial mRNA.
  Biochemistry, 39, 5623-5629.  
11092840 W.B.Watt, and A.M.Dean (2000).
Molecular-functional studies of adaptive genetic variation in prokaryotes and eukaryotes.
  Annu Rev Genet, 34, 593-622.  
10461177 R.Chen (1999).
A general strategy for enzyme engineering.
  Trends Biotechnol, 17, 344-345.  
15012211 W.H.Campbell (1999).
NITRATE REDUCTASE STRUCTURE, FUNCTION AND REGULATION: Bridging the Gap between Biochemistry and Physiology.
  Annu Rev Plant Physiol Plant Mol Biol, 50, 277-303.  
10477256 Y.Xu, G.Bhargava, H.Wu, G.Loeber, and L.Tong (1999).
Crystal structure of human mitochondrial NAD(P)+-dependent malic enzyme: a new class of oxidative decarboxylases.
  Structure, 7, R877-R889.  
9667915 J.L.Harris, and C.S.Craik (1998).
Engineering enzyme specificity.
  Curr Opin Chem Biol, 2, 127-132.  
9211842 A.D.Mesecar, B.L.Stoddard, and D.E.Koshland (1997).
Orbital steering in the catalytic power of enzymes: small structural changes with large catalytic consequences.
  Science, 277, 202-206.
PDB codes: 1ai2 1ai3
9325279 L.M.Watkins, H.J.Mahoney, J.K.McCulloch, and F.M.Raushel (1997).
Augmented hydrolysis of diisopropyl fluorophosphate in engineered mutants of phosphotriesterase.
  J Biol Chem, 272, 25596-25601.  
9428712 R.Chen, A.F.Greer, and A.M.Dean (1997).
Structural constraints in protein engineering--the coenzyme specificity of Escherichia coli isocitrate dehydrogenase.
  Eur J Biochem, 250, 578-582.  
9268311 W.N.Zhao, and L.McAlister-Henn (1997).
Affinity purification and kinetic analysis of mutant forms of yeast NAD+-specific isocitrate dehydrogenase.
  J Biol Chem, 272, 21811-21817.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.