PDBsum entry 1ocv

Ketosteroid isomerase

PDB id: 1ocv

Contents

Protein chains

125 a.a. *

Waters ×357

* Residue conservation analysis

PDB id:

1ocv

Name:

Ketosteroid isomerase

Title:

The f116w mutant structure of ketosteroid isomerase from comamonas testosteroni

Structure:

Steroid delta-isomerase. Chain: a, b, c, d. Synonym: ketosteroid isomerase. Engineered: yes. Mutation: yes

Source:

Comamonas testosteroni. Organism_taxid: 285. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Biol. unit:

Dimer (from PDB file)

Resolution:

2.00Å R-factor: 0.229 R-free: 0.282

Authors:

Y.S.Yun,T.-H.Lee,S.Shin

Key ref:

Y.S.Yun et al. (2003). Origin of the different pH activity profile in two homologous ketosteroid isomerases. J Biol Chem, 278, 28229-28236. PubMed id: 12734184 DOI: 10.1074/jbc.M302166200

Date:

11-Feb-03 Release date: 24-Jul-03

Protein chains

P00947  (SDIS_COMTE) -  Steroid Delta-isomerase from Comamonas testosteroni

Seq: 125 a.a.

Struc: 125 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.5.3.3.1 - steroid Delta-isomerase.
• Reaction: a 3-oxo-Delta₅-steroid = a 3-oxo-Delta₄-steroid

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

DOI no: 10.1074/jbc.M302166200

J Biol Chem 278:28229-28236 (2003)

PubMed id: 12734184

Origin of the different pH activity profile in two homologous ketosteroid isomerases.

Y.S.Yun, T.H.Lee, G.H.Nam, d.o. .S.Jang, S.Shin, B.H.Oh, K.Y.Choi.

ABSTRACT

Two homologous Delta5-3-ketosteroid isomerases from Comamonas testosteroni (TI-WT) and Pseudomonas putida biotype B (PI-WT) exhibit different pH activity profiles. TI-WT loses activity below pH 5.0 due to the protonation of the conserved catalytic base, Asp-38, while PI-WT does not. Based on the structural analysis of PI-WT, the critical catalytic base, Asp-38, was found to form a hydrogen bond with the indole ring NH of Trp-116, which is homologously replaced with Phe-116 in TI-WT. To investigate the role of Trp-116, we prepared the F116W mutant of TI-WT (TI-F116W) and the W116F mutant of PI-WT (PI-W116F) and compared kinetic parameters of those mutants at different pH levels. PI-W116F exhibited significantly decreased catalytic activity at acidic pH like TI-WT, whereas TI-F116W maintained catalytic activity at acidic pH like PI-WT and increased the kcat/Km value by 2.5- to 4.7-fold compared with TI-WT at pH 3.8. The crystal structure of TI-F116W clearly showed that the indole ring NH of Trp-116 could form a hydrogen bond with the carboxyl oxygen of Asp-38 like that of PI-WT. The present results demonstrate that the activities of both PI-WT and TI-F116W at low pH were maintained by a tryptophan, which was able not only to lower the pKa value of the catalytic base but also to increase the substrate affinity. This is one example of the strategy nature can adopt to evolve the diversity of the catalytic function in the enzymes. Our results provide insight into deciphering the molecular evolution of the enzyme and creating novel enzymes by protein engineering.

Selected figure(s)

Figure 2.
FIG. 2. pH dependence of kinetic parameters of WT and mutant KSIs. The lines represent nonlinear least-squares fits of the data to Equations 1 and 2 to obtain the pK[E] and pK[ES] values, respectively, as listed in Table I. 5,10-EST was used as a substrate for graphs C and D, and for PI-WT in graphs A and B. 5-AND was used as a substrate for graphs E and F, and for PI-W116F in graphs A and B.

Figure 4.
FIG. 4. Stereoview of the catalytic base of TI-F116W with 2F[o]-F[c]-simulated annealing omit electron density map contoured at 1.0 . Residues Trp-116 and Asp-38, which were omitted from the model, display clear electron density. A hydrogen bond between Asp-38 and Trp-116 is represented by a dashed line. The figure was drawn by using the program BobScript and rendered using Raster3D.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2003, 278, 28229-28236) copyright 2003.

Figures were selected by an automated process.