PDBsum entry 1w01

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Isomerase PDB id
Jmol PyMol
Protein chains
125 a.a. *
Waters ×38
* Residue conservation analysis
PDB id:
Name: Isomerase
Title: Crystal structure of mutant enzyme y57f/d103l of ketosteroid isomerase from pseudomonas putida biotype b
Structure: Steroid delta-isomerase. Chain: a, b. Synonym: delta-5-3-ketosteroid isomerase. Engineered: yes. Mutation: yes
Source: Pseudomonas putida. Organism_taxid: 303. Expressed in: escherichia coli. Expression_system_taxid: 469008.
Biol. unit: Dimer (from PDB file)
2.20Å     R-factor:   0.219     R-free:   0.268
Authors: D.S.Jang,H.J.Cha,K.Y.Choi
Key ref: D.S.Jang et al. (2004). Structural double-mutant cycle analysis of a hydrogen bond network in ketosteroid isomerase from Pseudomonas putida biotype B. Biochem J, 382, 967-973. PubMed id: 15228388
30-May-04     Release date:   08-Jul-04    
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Protein chains
Pfam   ArchSchema ?
P07445  (SDIS_PSEPU) -  Steroid Delta-isomerase
131 a.a.
125 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 2 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.  - Steroid Delta-isomerase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: A 3-oxo-Delta5-steroid = a 3-oxo-Delta4-steroid
= 3-oxo-Delta(4)-steroid
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     lipid metabolic process   2 terms 
  Biochemical function     isomerase activity     2 terms  


    Added reference    
Biochem J 382:967-973 (2004)
PubMed id: 15228388  
Structural double-mutant cycle analysis of a hydrogen bond network in ketosteroid isomerase from Pseudomonas putida biotype B.
D.S.Jang, H.J.Cha, S.S.Cha, B.H.Hong, N.C.Ha, J.Y.Lee, B.H.Oh, H.S.Lee, K.Y.Choi.
KSI (ketosteroid isomerase) catalyses an allylic isomerization reaction at a diffusion-controlled rate. A hydrogen bond network, Asp(99).Water(504).Tyr(14).Tyr(55).Tyr(30), connects two critical catalytic residues, Tyr(14) and Asp(99), with Tyr(30), Tyr(55) and a water molecule in the highly apolar active site of the Pseudomonas putida KSI. In order to characterize the interactions among these amino acids in the hydrogen bond network of KSI, double-mutant cycle analysis was performed, and the crystal structure of each mutant protein within the cycle was determined respectively to interpret the coupling energy. The DeltaDeltaG(o) values of Y14F/D99L (Tyr(14)-->Phe/Asp(99)-->Leu) KSI, 25.5 kJ/mol for catalysis and 28.9 kJ/mol for stability, were smaller than the sums (i.e. 29.7 kJ/mol for catalysis and 34.3 kJ/mol for stability) for single mutant KSIs respectively, indicating that the effect of the Y14F/D99L mutation was partially additive for both catalysis and stability. The partially additive effect of the Y14F/D99L mutation suggests that Tyr(14) and Asp(99) should interact positively for the stabilization of the transition state during the catalysis. The crystal structure of Y14F/D99L KSI indicated that the Y14F/D99L mutation increased the hydrophobic interaction while disrupting the hydrogen bond network. The DeltaDeltaG(o) values of both Y30F/D99L and Y55F/D99L KSIs for the catalysis and stability were larger than the sum of single mutants, suggesting that either Tyr(30) and Asp(99) or Tyr(55) and Asp(99) should interact negatively for the catalysis and stability. These synergistic effects of both Y30F/D99L and Y55F/D99L mutations resulted from the disruption of the hydrogen bond network. The synergistic effect of the Y55F/D99L mutation was larger than that of the Y30F/D99L mutation, since the former mutation impaired the proper positioning of a critical catalytic residue, Tyr(14), involved in the catalysis of KSI. The present study can provide insight into interpreting the coupling energy measured by double-mutant cycle analysis based on the crystal structures of the wild-type and mutant proteins.

Literature references that cite this PDB file's key reference

  PubMed id Reference
19828079 F.Chiappori, P.D'Ursi, I.Merelli, L.Milanesi, and E.Rovida (2009).
In silico saturation mutagenesis and docking screening for the analysis of protein-ligand interaction: the Endothelial Protein C Receptor case study.
  BMC Bioinformatics, 10, S3.  
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