PDBsum entry: 1ogz

Isomerase

PDB id: 1ogz

Contents

Protein chain

125 a.a. *

Ligands

EQU

Waters ×93

* Residue conservation analysis

PDB id:

1ogz

Name:

Isomerase

Title:

Crystal structure of 5-3-ketosteroid isomerase mutants p39a complexed with equilenin from pseudomonas testosteroni

Structure:

Steroid delta-isomerase. Chain: a. Synonym: delta-5-3-ketosteroid isomerase, ketosteroid isomerase. Engineered: yes. Mutation: yes. Other_details: d-equilenin binding at hydrophobic pocket.

Source:

Comamonas testosteroni. Organism_taxid: 285. Expressed in: escherichia coli. Expression_system_taxid: 511693.

Biol. unit:

Dimer (from PDB file)

Resolution:

2.30Å R-factor: 0.235 R-free: 0.309

Authors:

G.H.Nam,S.-S.Cha,Y.S.Yun,Y.H.Oh,B.H.Hong,H.-S.Lee,K.Y.Choi

Key ref:

G.H.Nam et al. (2003). The conserved cis-Pro39 residue plays a crucial role in the proper positioning of the catalytic base Asp38 in ketosteroid isomerase from Comamonas testosteroni. Biochem J, 375, 297-305. PubMed id: 12852789 DOI: 10.1042/BJ20030263

Date:

20-May-03 Release date: 04-Sep-03

Protein chain

P00947  (SDIS_COMTE) -  Steroid Delta-isomerase

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

3-oxo-Delta(5)-steroid (Bound ligand (Het Group name = EQU) matches with 90.00% similarity) = 3-oxo-Delta(4)-steroid

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

 Gene Ontology (GO) functional annotation 

• Cellular component: intracellular (1 term)
• Biological process: transport (3 terms)
• Biochemical function: isomerase activity (2 terms)

Added reference

DOI no: 10.1042/BJ20030263

Biochem J 375:297-305 (2003)

PubMed id: 12852789

The conserved cis-Pro39 residue plays a crucial role in the proper positioning of the catalytic base Asp38 in ketosteroid isomerase from Comamonas testosteroni.

G.H.Nam, S.S.Cha, Y.S.Yun, Y.H.Oh, B.H.Hong, H.S.Lee, K.Y.Choi.

ABSTRACT

KSI (ketosteroid isomerase) from Comamonas testosteroni is a homodimeric enzyme that catalyses the allylic isomerization of Delta5-3-ketosteroids to their conjugated Delta4-isomers at a reaction rate equivalent to the diffusion-controlled limit. Based on the structural analysis of KSI at a high resolution, the conserved cis-Pro39 residue was proposed to be involved in the proper positioning of Asp38, a critical catalytic residue, since the residue was found not only to be structurally associated with Asp38, but also to confer a structural rigidity on the local active-site geometry consisting of Asp38, Pro39, Val40, Gly41 and Ser42 at the flexible loop between b-strands B1 and B2. In order to investigate the structural role of the conserved cis-Pro39 residue near the active site of KSI, Pro39 was replaced with alanine or glycine. The free energy of activation for the P39A and P39G mutants increased by 10.5 and 16.7 kJ/mol (2.5 and 4.0 kcal/mol) respectively, while DG(U)H2O (the free-energy change for unfolding in the absence of urea at 25.00+/-0.02 degrees C) decreased by 31.0 and 35.6 kJ/mol (7.4 and 8.5 kcal/mol) respectively, compared with the wild-type enzyme. The crystal structure of the P39A mutant in complex with d-equilenin [d-1,3,5(10),6,8-estrapentaen-3-ol-17-one], a reaction intermediate analogue, determined at 2.3 A (0.23 nm) resolution revealed that the P39A mutation significantly disrupted the proper orientations of both d-equilenin and Asp38, as well as the local active-site geometry near Asp38, which resulted in substantial decreases in the activity and stability of KSI. Upon binding 1-anilinonaphthalene-8-sulphonic acid, the fluorescence intensities of the P39A and P39G mutants were increased drastically, with maximum wavelengths blue-shifted upon binding, indicating that the mutations might alter the hydrophobic active site of KSI. Taken together, our results demonstrate that the conserved cis-Pro39 residue plays a crucial role in the proper positioning of the critical catalytic base Asp38 and in the structural integrity of the active site in KSI.