Maintenance of alpha-helical structures by phenyl rings in the active-site tyrosine triad contributes to catalysis and stability of ketosteroid isomerase from Pseudomonas putida biotype B.


Abstract

Ketosteroid isomerase (KSI) from Pseudomonas putida biotype B is a homodimeric enzyme catalyzing an allylic rearrangement of Delta5-3-ketosteroids at rates comparable with the diffusion-controlled limit. The tyrosine triad (Tyr14.Tyr55.Tyr30) forming a hydrogen-bond network in the apolar active site of KSI has been characterized in an effort to identify the roles of the phenyl rings in catalysis, stability, and unfolding of the enzyme. The replacement of Tyr14, a catalytic residue, with serine resulted in a 33-fold decrease of kcat, while the replacements of Tyr30 and Tyr55 with serine decreased kcat by 4- and 51-fold, respectively. The large decrease of kcat for Y55S could be due to the structural perturbation of alpha-helix A3, which results in the reorientation of the active-site residues as judged by the crystal structure of Y55S determined at 2.2 A resolution. Consistent with the analysis of the Y55S crystal structure, the far-UV circular dichroism spectra of Y14S, Y30S, and Y55S indicated that the elimination of the phenyl ring of the tyrosine reduced significantly the content of alpha-helices. Urea-induced equilibrium unfolding experiments revealed that the DeltaG(U)H2O values of Y14S, Y30S, and Y55S were significantly decreased by 11.9, 13.7, and 9.5 kcal/mol, respectively, as compared with that of the wild type. A characterization of the unfolding kinetics based on PhiU-value analysis indicates that the interactions mediated by the tyrosine triad in the native state are very resistant to unfolding. Taken together, our results demonstrate that the internal packing by the phenyl rings in the active-site tyrosine triad contributes to the conformational stability and catalytic activity of KSI by maintaining the structural integrity of the alpha-helices. Study holds ProTherm entries: 12234, 12235, 12236, 12237 Extra Details: 1mM EDTA and 1mM DTT as additive. hydrogen-bond network; phenyl rings; unfolding kinetics;,structural integrity; alpha-helices

Submission Details

ID: 5ogtDmf43

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:43 p.m.

Version: 1

Publication Details
Nam GH;Jang DS;Cha SS;Lee TH;Kim DH;Hong BH;Yun YS;Oh BH;Choi KY,Biochemistry (2001) Maintenance of alpha-helical structures by phenyl rings in the active-site tyrosine triad contributes to catalysis and stability of ketosteroid isomerase from Pseudomonas putida biotype B. PMID:11695900
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
6C1X 2018-07-25 1.05 Crystal Structure of Ketosteroid Isomerase D40N/D103N mutant from Pseudomonas Putida (pKSI) bound to 3,4-dinitrophenol
6C1J 2018-07-25 1.06 Crystal Structure of Ketosteroid Isomerase Y32F/Y57F/D40N mutant from Pseudomonas Putida (pKSI) bound to 3,4-dinitrophenol
6F54 2018-05-23 1.08 CRYSTAL STRUCTURE OF KETOSTEROID ISOMERASE TRIPLE VARIANT V88I/L99VD103S
6C17 2018-07-25 1.1 Crystal Structure of Ketosteroid Isomerase D40N mutant from Pseudomonas Putida (pKSI) bound to 3,4-dinitrophenol
1OH0 2003-06-04 1.1 CRYSTAL STRUCTURE OF KETOSTEROID ISOMERASE COMPLEXED WITH EQUILENIN
5KP1 2016-09-07 1.22 Crystal Structure of Ketosteroid Isomerase from Pseudomonas putida (pKSI) bound to Equilenin; D40N, Y16(Cl-Y)
3CPO 2008-09-23 1.24 Crystal structure of ketosteroid isomerase D40N with bound 2-fluorophenol
2PZV 2007-06-12 1.25 Crystal Structure of Ketosteroid Isomerase D40N from Pseudomonas Putida (pksi) with bound Phenol
3SED 2011-08-03 1.3 Crystal Structure of Ketosteroid Isomerase Variant M105A from Pseudomonos putida
3VGN 2012-08-22 1.3 Crystal Structure of Ketosteroid Isomerase D40N from Pseudomonas putida (pKSI) with bound 3-fluoro-4-nitrophenol
3FZW 2009-06-02 1.32 Crystal Structure of Ketosteroid Isomerase D40N-D103N from Pseudomonas putida (pKSI) with bound equilenin
5D82 2015-12-02 1.37 Crystal Structure of Ketosteroid Isomerase from Pseudomonas putida (pKSI); D40N, Y16(Cl-Y)
5D81 2015-12-02 1.39 Crystal Structure of Ketosteroid Isomerase from Pseudomonas putida (pKSI); D40N, Y57(Cl-Y)
3T8N 2011-11-23 1.47 Crystal structure of ketosteroid isomerase Y16AD103A from Pseudomonas putida
6F53 2018-05-23 1.49 CRYSTAL STRUCTURE OF KETOSTEROID ISOMERASE QUADRUPLE VARIANT V88I/L99V/D103S/V101A
3VSY 2013-02-27 1.5 High-resolution crystal structure of wild-type KSI in the apo form at neutral pH
2INX 2007-10-23 1.5 Crystal Structure of Ketosteroid Isomerase D40N from Pseudomonas putida (pKSI) with bound 2,6-difluorophenol
3RGR 2012-08-22 1.59 Crystal structure of ketosteroid isomerase M116A from Pseudomonas putida
5G2G 2016-07-13 1.6 Crystal structure of ketosteroid isomerase containing M116K mutation in the equilenin-bound form
3IPT 2010-01-12 1.63 Crystal Structure of Ketosteroid Isomerase Y16S/D40N from Pseudomonas putida with Bound Equilenin
5D83 2015-12-02 1.7 Crystal Structure of Ketosteroid Isomerase from Pseudomonas putida (pKSI); D40N, Y32(Cl-Y)
5KP3 2016-09-07 1.7 Crystal Structure of Ketosteroid Isomerase from Pseudomonas putida (pKSI) bound to Equilenin; D40N, Y57(Cl-Y)
3OWU 2011-09-21 1.7 Crystal Structure of Ketosteroid Isomerase D40N/C69S/C81S/C97S/F86C-CN from P. putida with Bound Equilenin
3OWS 2011-09-21 1.71 Crystal Structure of Ketosteroid Isomerase D40N/C69S/C81S/C97S/M116C-CN from P. putida with Bound Equilenin
5KP4 2016-09-07 1.71 Crystal Structure of Ketosteroid Isomerase from Pseudomonas putida (pKSI) bound to 19-nortestosterone
1EA2 2001-11-01 1.8 Pseudoreversion of the Catalytic Activity of Y14F by the Additional Tyrosine-to-Phenylalanine Substitution(s) in the Hydrogen Bond Network of Delta-5-3-Ketosteroid Isomerase from Pheudomonas putida Biotype B
4K1V 2013-07-31 1.8 Crystal structure of delta5-3-ketosteroid isomerase containing Y16F and Y57F mutations
3OXA 2011-09-21 1.89 Crystal Structure of Ketosteroid Isomerase D40N/C69S/C81S/C97S/M116C-CN from P. putida
1CQS 2003-06-17 1.9 CRYSTAL STRUCTURE OF D103E MUTANT WITH EQUILENINEOF KSI IN PSEUDOMONAS PUTIDA
1DMM 2000-05-23 1.9 CRYSTAL STRUCTURES OF MUTANT ENZYMES Y57F OF KETOSTEROID ISOMERASE FROM PSEUDOMONAS PUTIDA BIOTYPE B
1OHO 2005-03-31 1.9 CRYSTAL STRUCTURE OF KETOSTEROID ISOMERASE Y16F/D40N mutant COMPLEXED WITH EQUILENIN
1OPY 1998-04-08 1.9 KSI
6F4Y 2018-05-23 1.92 CRYSTAL STRUCTURE OF KETOSTEROID ISOMERASE VARIANT D103S
1E3V 2001-03-12 2.0 Crystal structure of ketosteroid isomerase from Psedomonas putida complexed with deoxycholate
1E97 2001-10-05 2.0 Crystal structure of ketosteroid isomerase from Pseudomonas putida ; triple mutant y16f/y32f/y57f
1OGX 2003-05-20 2.0 High Resolution Crystal Structure Of Ketosteroid Isomerase Mutant D40N(D38N, Ti Numbering) from Pseudomonas putida Complexed With Equilenin At 2.0 A Resolution.
3OX9 2011-09-21 2.0 Crystal Structure of Ketosteroid Isomerase D40N/C69S/C81S/C97S/F86C-CN from P. putida
4K1U 2013-07-31 2.0 Crystal structure of delta5-3-ketosteroid isomerase containing Y16F and Y32F mutations
6F50 2018-05-23 2.0 CRYSTAL STRUCTURE OF KETOSTEROID ISOMERASE DOUBLE VARIANT V88I/L99V
1DMN 2000-05-23 2.05 CRYSTAL STRUCTURE OF MUTANT ENZYME Y32F/Y57F OF KETOSTEROID ISOMERASE FROM PSEUDOMONAS PUTIDA BIOTYPE B
1GS3 2003-01-23 2.1 High resolution crystal structure of PI delta-5-3-Ketosteroid Isomerase mutants Y30F/Y55F/Y115F/D38N (Y32F/Y57F/Y119F/D40N, PI numbering)complexed with equilenin at 2.1 A resolution
5AI1 2015-05-20 2.1 Crystal structure of ketosteroid isomerase containing Y32F, D40N, Y57F and Y119F mutations in the equilenin-bound form
1W6Y 2005-04-14 2.1 crystal structure of a mutant W92A in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B
1DMQ 2000-05-23 2.15 CRYSTAL STRUCTURE OF MUTANT ENZYME Y32F OF KETOSTEROID ISOMERASE FROM PSEUDOMONAS PUTIDA BIOTYPE B
1W01 2004-07-08 2.2 Crystal structure of mutant enzyme Y57F/D103L of ketosteroid isomerase from Pseudomonas putida biotype B
1W00 2005-05-26 2.2 Crystal structure of mutant enzyme D103L of Ketosteroid Isomerase from Pseudomonas putida biotype B
1K41 2002-10-16 2.2 Crystal structure of KSI Y57S mutant
1VZZ 2004-07-08 2.3 CRYSTAL STRUCTURE OF MUTANT ENZYME Y32F/D103L OF KETOSTEROID ISOMERASE FROM PSEUDOMONAS PUTIDA BIOTYPE B
3OWY 2011-09-21 2.3 Crystal Structure of Ketosteroid Isomerase D40N/C69S/C81S/C97S/M105C-CN from P. putida with Bound Equilenin
1W02 2004-07-08 2.3 Crystal structure of mutant enzyme Y16F/D103L of ketosteroid isomerase from Pseudomonas putida biotype B
4CDL 2014-11-12 2.5 Crystal Structure of Retro-aldolase RA110.4-6 Complexed with Inhibitor 1-(6-methoxy-2-naphthalenyl)-1,3-butanedione
1E3R 2001-03-05 2.5 Crystal structure of ketosteroid isomerase mutant D40N (D38N TI numbering) from Pseudomonas putida complexed with androsten-3beta-ol-17-one
1C7H 2000-04-24 2.5 CRYSTAL STRUCTURE OF A MUTANT R75A IN KETOSTEROID ISOMERASE FROM PSEDOMONAS PUTIDA BIOTYPE B

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Steroid Delta-isomerase P07445 SDIS_PSEPU