1K41

Crystal structure of KSI Y57S mutant

1K41 の概要

• エントリーDOI: 10.2210/pdb1k41/pdb
• 分子名称: Ketosteroid Isomerase (2 entities in total)
• 機能のキーワード: ksi y57s helix, isomerase
• 由来する生物種: Pseudomonas putida
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 28944.81

構造登録者

Cha, S.S.,Oh, B.H.,Nam, G.H.,Jang, D.S.,Lee, T.H.,Choi, K.Y. (登録日: 2001-10-05, 公開日: 2002-10-16, 最終更新日: 2024-05-29)

主引用文献

Nam, G.H.,Jang, D.S.,Cha, S.S.,Lee, T.H.,Kim, D.H.,Hong, B.H.,Yun, Y.S.,Oh, B.H.,Choi, K.Y.
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
Biochemistry, 40:13529-13537, 2001

PubMed 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.

PubMed: 11695900
DOI: 10.1021/bi015547k

実験手法

X-RAY DIFFRACTION (2.2 Å)