1W02

Crystal structure of mutant enzyme Y16F/D103L of ketosteroid isomerase from Pseudomonas putida biotype B

Summary for 1W02

• Entry DOI: 10.2210/pdb1w02/pdb
• Related: 1C7H 1CQS 1DMM 1DMN 1DMQ 1E3R 1E3V 1E97 1EA2 1GS3 1K41 1OGX 1OH0 1OHO 1OPY 1VZZ 1W00 1W01
• Descriptor: STEROID DELTA-ISOMERASE (2 entities in total)
• Functional Keywords: isomerase, coneshell, closed barrel, curved b-sheet
• Biological source: PSEUDOMONAS PUTIDA
• Total number of polymer chains: 1
• Total formula weight: 14530.57

Authors

Jang, D.S.,Choi, K.Y. (deposition date: 2004-05-30, release date: 2004-07-08, Last modification date: 2024-05-08)

Primary citation

Jang, D.S.,Cha, H.J.,Cha, S.S.,Hong, B.H.,Ha, N.C.,Lee, J.Y.,Oh, B.H.,Lee, H.S.,Choi, K.Y.
Structural Double-Mutant Cycle Analysis of a Hydrogen Bond Network in Ketosteroid Isomerase from Pseudomonas Putida Biotype B.
Biochem.J., 382:967-, 2004

PubMed Abstract: 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.

PubMed: 15228388
DOI: 10.1042/BJ20031871

Experimental method

X-RAY DIFFRACTION (2.3 Å)