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
1C7H 2000-02-19T00:00:00+0000 2.5 CRYSTAL STRUCTURE OF A MUTANT R75A IN KETOSTEROID ISOMERASE FROM PSEDOMONAS PUTIDA BIOTYPE B
1CQS 1999-08-11T00:00:00+0000 1.9 CRYSTAL STRUCTURE OF D103E MUTANT WITH EQUILENINEOF KSI IN PSEUDOMONAS PUTIDA
1DMM 1999-12-14T00:00:00+0000 1.9 CRYSTAL STRUCTURES OF MUTANT ENZYMES Y57F OF KETOSTEROID ISOMERASE FROM PSEUDOMONAS PUTIDA BIOTYPE B
1DMN 1999-12-14T00:00:00+0000 2.05 CRYSTAL STRUCTURE OF MUTANT ENZYME Y32F/Y57F OF KETOSTEROID ISOMERASE FROM PSEUDOMONAS PUTIDA BIOTYPE B
1DMQ 1999-12-14T00:00:00+0000 2.15 CRYSTAL STRUCTURE OF MUTANT ENZYME Y32F OF KETOSTEROID ISOMERASE FROM PSEUDOMONAS PUTIDA BIOTYPE B
1E3R 2000-06-22T00:00:00+0000 2.5 Crystal structure of ketosteroid isomerase mutant D40N (D38N TI numbering) from Pseudomonas putida complexed with androsten-3beta-ol-17-one
1E3V 2000-06-24T00:00:00+0000 2.0 Crystal structure of ketosteroid isomerase from Psedomonas putida complexed with deoxycholate
1E97 2000-10-10T00:00:00+0000 2.0 Crystal structure of ketosteroid isomerase from Pseudomonas putida ; triple mutant y16f/y32f/y57f
1EA2 2000-11-03T00:00:00+0000 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
1GS3 2001-12-27T00:00:00+0000 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

Relevant UniProtKB Entries

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