Contribution of the hydrogen-bond network involving a tyrosine triad in the active site to the structure and function of a highly proficient ketosteroid isomerase from Pseudomonas putida biotype B.


Abstract

Delta(5)-3-Ketosteroid isomerase from Pseudomonas putida biotype B is one of the most proficient enzymes catalyzing an allylic isomerization reaction at rates comparable to the diffusion limit. The hydrogen-bond network (Asp99... Wat504...Tyr14...Tyr55...Tyr30) which links the two catalytic residues, Tyr14 and Asp99, to Tyr30, Tyr55, and a water molecule in the highly apolar active site has been characterized in an effort to identify its roles in function and stability. The DeltaG(U)(H2O) determined from equilibrium unfolding experiments reveals that the elimination of the hydroxyl group of Tyr14 or Tyr55 or the replacement of Asp99 with leucine results in a loss of conformational stability of 3.5-4.4 kcal/mol, suggesting that the hydrogen bonds of Tyr14, Tyr55, and Asp99 contribute significantly to stability. While decreasing the stability by about 6.5-7.9 kcal/mol, the Y55F/D99L or Y30F/D99L double mutation also reduced activity significantly, exhibiting a synergistic effect on k(cat) relative to the respective single mutations. These results indicate that the hydrogen-bond network is important for both stability and function. Additionally, they suggest that Tyr14 cannot function efficiently alone without additional support from the hydrogen bonds of Tyr55 and Asp99. The crystal structure of Y55F as determined at 1.9 A resolution shows that Tyr14 OH undergoes an alteration in orientation to form a new hydrogen bond with Tyr30. This observation supports the role of Tyr55 OH in positioning Tyr14 properly to optimize the hydrogen bond between Tyr14 and C3-O of the steroid substrate. No significant structural changes were observed in the crystal structures of Y30F and Y30F/Y55F, which allowed us to estimate approximately the interaction energies mediated by the hydrogen bonds Tyr30...Tyr55 and Tyr14...Tyr55. Taken together, our results demonstrate that the hydrogen-bond network provides the structural support that is needed for the enzyme to maintain the active-site geometry optimized for both function and stability. Study holds ProTherm entries: 7964, 7965, 7966, 7967, 7968, 7969, 7970, 7971 Extra Details: additive : EDTA(0.5 mM),dithiothreitol(1 mM) was added in the experiment isomerization reaction; hydrogen-bond network; conformational,stability; synergistic effect

Submission Details

ID: QUmDCh8y3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:35 p.m.

Version: 1

Publication Details
Kim DH;Jang DS;Nam GH;Choi G;Kim JS;Ha NC;Kim MS;Oh BH;Choi KY,Biochemistry (2000) Contribution of the hydrogen-bond network involving a tyrosine triad in the active site to the structure and function of a highly proficient ketosteroid isomerase from Pseudomonas putida biotype B. PMID:10769113
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
1OH0 2003-06-04 1.1 CRYSTAL STRUCTURE OF KETOSTEROID ISOMERASE COMPLEXED WITH EQUILENIN
6C17 2018-07-25 1.1 Crystal Structure of Ketosteroid Isomerase D40N mutant from Pseudomonas Putida (pKSI) bound to 3,4-dinitrophenol
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
2INX 2007-10-23 1.5 Crystal Structure of Ketosteroid Isomerase D40N from Pseudomonas putida (pKSI) with bound 2,6-difluorophenol
3VSY 2013-02-27 1.5 High-resolution crystal structure of wild-type KSI in the apo form at neutral pH
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
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
5D83 2015-12-02 1.7 Crystal Structure of Ketosteroid Isomerase from Pseudomonas putida (pKSI); D40N, Y32(Cl-Y)
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
4K1V 2013-07-31 1.8 Crystal structure of delta5-3-ketosteroid isomerase containing Y16F and Y57F mutations
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
3OXA 2011-09-21 1.89 Crystal Structure of Ketosteroid Isomerase D40N/C69S/C81S/C97S/M116C-CN from P. putida
1OPY 1998-04-08 1.9 KSI
1OHO 2005-03-31 1.9 CRYSTAL STRUCTURE OF KETOSTEROID ISOMERASE Y16F/D40N mutant COMPLEXED WITH EQUILENIN
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
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
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.
1E97 2001-10-05 2.0 Crystal structure of ketosteroid isomerase from Pseudomonas putida ; triple mutant y16f/y32f/y57f
4K1U 2013-07-31 2.0 Crystal structure of delta5-3-ketosteroid isomerase containing Y16F and Y32F mutations
3OX9 2011-09-21 2.0 Crystal Structure of Ketosteroid Isomerase D40N/C69S/C81S/C97S/F86C-CN from P. putida
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
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
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
1DMQ 2000-05-23 2.15 CRYSTAL STRUCTURE OF MUTANT ENZYME Y32F 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
1W01 2004-07-08 2.2 Crystal structure of mutant enzyme Y57F/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
1VZZ 2004-07-08 2.3 CRYSTAL STRUCTURE OF MUTANT ENZYME Y32F/D103L OF KETOSTEROID ISOMERASE FROM PSEUDOMONAS PUTIDA BIOTYPE B
1C7H 2000-04-24 2.5 CRYSTAL STRUCTURE OF A MUTANT R75A IN KETOSTEROID ISOMERASE FROM PSEDOMONAS PUTIDA BIOTYPE B
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
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

Relevant UniProtKB Entries

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