1w02
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CRYSTAL STRUCTURE OF MUTANT ENZYME Y16F/D103L OF KETOSTEROID ISOMERASE FROM PSEUDOMONAS PUTIDA BIOTYPE B
OverviewOverview
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.
About this StructureAbout this Structure
1W02 is a Single protein structure of sequence from Pseudomonas putida. Active as Steroid Delta-isomerase, with EC number 5.3.3.1 Full crystallographic information is available from OCA.
ReferenceReference
Structural double-mutant cycle analysis of a hydrogen bond network in ketosteroid isomerase from Pseudomonas putida biotype B., Jang DS, Cha HJ, Cha SS, Hong BH, Ha NC, Lee JY, Oh BH, Lee HS, Choi KY, Biochem J. 2004 Sep 15;382(Pt 3):967-73. PMID:15228388
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