Equilibrium and kinetic analysis of folding of ketosteroid isomerase from Comamonas testosteroni.


Abstract

Equilibrium and kinetic analyses have been carried out to elucidate the folding mechanism of homodimeric ketosteroid isomerase (KSI) from Comamonas testosteroni. The folding of KSI was reversible since the activity as well as the fluorescence and CD spectra was almost completely recovered after refolding. The equilibrium unfolding transitions monitored by fluorescence and CD measurements were almost coincident with each other, and the transition midpoint increased with increasing protein concentration. This suggests that the KSI folding follows a simple two-state mechanism consisting of native dimer and unfolded monomer without any thermodynamically stable intermediates. Sedimentation equilibrium analysis and size-exclusion chromatography of KSI at different urea concentrations supported the two-state model without any evidence of folded monomeric intermediates. Consistent with the two-state model, (1)H-(15)N HSQC spectra obtained for KSI in the unfolding transition region could be reproduced by a simple addition of the spectra of the native and the unfolded KSI. The KSI refolding kinetics as monitored by fluorescence intensity could be described as a fast first-order process followed by a second-order and a subsequent slow first-order processes with rate constants of 60 s(-)(1), 5.4 x 10(4) M(-)(1).s(-)(1), and 0.017 s(-)(1), respectively, at 0.62 M urea, suggesting that there may be a monomeric folding intermediate. After a burst phase that accounts for >83% of the total amplitude, the negative molar ellipticity at 225 nm increased slowly in a single phase at a rate comparable to that of the bimolecular intermediate step. The kinetics of activity recovery from the denatured state were markedly dependent upon the protein concentration, implying that the monomers are not fully active. Taken together, our results demonstrate that the dimerization induces KSI to fold into the complete structure and is crucial for maintaining the tertiary structure to perform efficient catalysis. Study holds ProTherm entries: 9649 Extra Details: additive : EDTA(0.5 mM), two-state mechanism; refolding kinetics; dimerization;,tertiary structure; catalysis

Submission Details

ID: XXCRbUsH3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:38 p.m.

Version: 1

Publication Details
Kim DH;Jang DS;Nam GH;Yun S;Cho JH;Choi G;Lee HC;Choi KY,Biochemistry (2000) Equilibrium and kinetic analysis of folding of ketosteroid isomerase from Comamonas testosteroni. PMID:11041875
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1BUQ 1998-09-04T00:00:00+0000 0 SOLUTION STRUCTURE OF DELTA-5-3-KETOSTEROID ISOMERASE COMPLEXED WITH THE STEROID 19-NORTESTOSTERONE-HEMISUCCINATE
1ISK 1997-03-12T00:00:00+0000 0 3-OXO-DELTA5-STEROID ISOMERASE, NMR, 20 STRUCTURES
1OCV 2003-02-11T00:00:00+0000 2.0 the F116W mutant structure of ketosteroid isomerase from Comamonas testosteroni
1OGZ 2003-05-20T00:00:00+0000 2.3 Crystal Structure Of 5-3-Ketosteroid Isomerase Mutants P39A Complexed With Equilenin From Pseudomonas Testosteroni
1OHP 2003-05-29T00:00:00+0000 1.53 CRYSTAL STRUCTURE OF 5-3-KETOSTEROID ISOMERASE MUTANT D38N FROM PSEUDOMONAS TESTOSTERONI COMPLEXED WITH 5ALPHA-ESTRAN-3,17-DIONE
1OHS 2003-05-30T00:00:00+0000 1.7 CRYSTAL STRUCTURE OF 5-3-KETOSTEROID ISOMERASE MUTANT Y14F/D38N FROM PSEUDOMONAS TESTOSTERONI COMPLEXED WITH ANDROSTANEDIONE
1QJG 1999-06-24T00:00:00+0000 2.3 Crystal structure of delta5-3-ketosteroid isomerase from Pseudomonas testosteroni in complex with equilenin
3M8C 2010-03-17T00:00:00+0000 2.1 Crystal Structure of Ketosteroid Isomerase D99N from Pseudomonas Testosteroni (tKSI) with Equilenin Bound
3MHE 2010-04-07T00:00:00+0000 1.72 Crystal Structure of Ketosteroid Isomerase P39A from Pseudomonas Testosteroni (tKSI)
3MKI 2010-04-14T00:00:00+0000 2.0 Crystal Structure of Ketosteroid Isomerase D38ED99N from Pseudomonas Testosteroni (tKSI)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Steroid Delta-isomerase P00947 SDIS_COMTE