Contribution of conserved amino acids at the dimeric interface to the conformational stability and the structural integrity of the active site in ketosteroid isomerase from Pseudomonas putida biotype B.

Ketosteroid isomerase (KSI) from Pseudomonas putida biotype B is a homodimeric enzyme catalyzing an allylic isomerization of Delta(5)-3-ketosteroids at a rate of the diffusion-controlled limit. The dimeric interactions mediated by Arg72, Glu118, and Asn120, which are conserved in the homologous KSIs, have been characterized in an effort to investigate the roles of the conserved interface residues in stability, function and structure of the enzyme. The interface residues were replaced with alanine to generate the interface mutants R72A, E118A, N120A and E118A/N120A.

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

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.

Origin of the different pH activity profile in two homologous ketosteroid isomerases.

Two homologous Delta5-3-ketosteroid isomerases from Comamonas testosteroni (TI-WT) and Pseudomonas putida biotype B (PI-WT) exhibit different pH activity profiles. TI-WT loses activity below pH 5.0 due to the protonation of the conserved catalytic base, Asp-38, while PI-WT does not.

Association of human tumor necrosis factor-related apoptosis inducing ligand with membrane upon acidification.

Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) has been known to induce tumor-specific apoptosis and to share the structural and functional characteristics with the proteins of TNF family. Recently, the crystal structure of human TRAIL showed that TRAIL is a homotrimeric protein whose subunits contain mainly beta-sheets. We characterized the structural changes of recombinant human TRAIL induced by acidification and the biological implication of the structural characteristics at acidic pH in the interaction with the lipid bilayer.