1DMM

CRYSTAL STRUCTURES OF MUTANT ENZYMES Y57F OF KETOSTEROID ISOMERASE FROM PSEUDOMONAS PUTIDA BIOTYPE B

1DMM の概要

• エントリーDOI: 10.2210/pdb1dmm/pdb
• 関連するPDBエントリー: 1DMM 1DMN 1DMQ 1QJG 8CHO
• 分子名称: STEROID DELTA-ISOMERASE (2 entities in total)
• 機能のキーワード: coneshell, closed barrel, curved b-sheet, isomerase
• 由来する生物種: Pseudomonas putida
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 14532.50

構造登録者

Kim, D.H.,Jang, D.S.,Nam, G.H.,Oh, B.H.,Choi, K.Y. (登録日: 1999-12-14, 公開日: 2000-05-23, 最終更新日: 2024-02-07)

主引用文献

Kim, D.H.,Jang, D.S.,Nam, G.H.,Choi, G.,Kim, J.S.,Ha, N.C.,Kim, M.S.,Oh, B.H.,Choi, K.Y.
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.
Biochemistry, 39:4581-4589, 2000

PubMed 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.

PubMed: 10769113
DOI: 10.1021/bi992119u

実験手法

X-RAY DIFFRACTION (1.9 Å)