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 Biotyp

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• 作者：Do-Hyung Kim ; Do Soo Jang ; Gyu Hyun Nam ; Gildon Choi ; Jeong-Sun Kim ; Nam-Chul Ha ; Min-Sung Kim ; Byung-Ha Oh ; and Kwan Yong Choi
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：April 25, 2000
• 年：2000
• 卷：39
• 期：16
• 页码：4581 - 4589
• 全文大小：114K
• 年卷期：v.39,no.16(April 25, 2000)
• ISSN：1520-4995

文摘

⁵-3-Ketosteroid isomerase from Pseudomonas putida biotype B is one of the most proficientenzymes catalyzing an allylic isomerization reaction at rates comparable to the diffusion limit. Thehydrogen-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 beencharacterized in an effort to identify its roles in function and stability. The G_U^H₂O determined fromequilibrium unfolding experiments reveals that the elimination of the hydroxyl group of Tyr14 or Tyr55or 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 mutationalso reduced activity significantly, exhibiting a synergistic effect on k_cat relative to the respective singlemutations. 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 fromthe hydrogen bonds of Tyr55 and Asp99. The crystal structure of Y55F as determined at 1.9 Å resolutionshows 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 hydrogenbond between Tyr14 and C3-O of the steroid substrate. No significant structural changes were observedin the crystal structures of Y30F and Y30F/Y55F, which allowed us to estimate approximately theinteraction 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 forthe enzyme to maintain the active-site geometry optimized for both function and stability.