利用飽和定點突變方法探討酵母菌氧化鯊烯環化酵素內Tyr707 和Tyr710 對反應過程中B 環形成的影響

Abstract

(3S)-2,3-氧化鯊烯((3S)-2,3-oxidosqulaene)可經由不同的環化酵素催化而產生各式各樣的三萜類化合物，這複雜的環化反應在這半世紀以來一直令有機及生物化學家為之著迷。在酵母菌及哺乳類動物中，氧化鯊烯環化酵素(oxidosqualene-lanosterol cyclase, ERG7)催化(3S)-2,3-氧化鯊烯產生具有四個環的羊毛硬脂醇(lanosterol)，透過所謂的”椅形-船形-椅形” (chair-boat-chair)的折疊方式。這個複雜的環化/重組反應機制包括一開始氧化鯊烯的環氧基被質子化而斷裂，接著由碳陽離子-π電子作用 (cationic-π interaction)所引導的四個環的環化反應和一連串氫化基及甲基的重排，以及最後具有高度專一性的去質子化步驟。在此我們利用飽和定點突變的方法，來探討酵母菌的氧化鯊烯環化酵素內Tyr707以及Tyr710這兩個被高度保留的芳香性胺基酸，在酵素催化反應的過程中所扮演的角色。經由產物的分析，我們在ERG7Y707X的突變株中，分離出先前從未被發表過的雙環新產物(9R,10S)-polypoda-8(26),13E,17E,21-tetraen-3β-ol，以及其他因改變最後的去質子化位置而造成的產物。因此我們推測Tyr707在環化機制中可能是扮演穩定雙環C-8碳陽離子以及最終的羊毛硬脂醇C-8/C-9碳陽離子中間物的角色。將這個位置的胺基酸替換成其他胺基酸則會干擾整個環化/重組反應的過程，因而導致環化不完全的雙環產物以及其他改變最後去質子化位置的產物產生。另外在ERG7Y710X方面，由飽和定點突變分析顯示，在某幾個突變點中產生了羊毛硬脂醇及兩個因改變最後的質子化位置而造成的產物，parkeol和9β-lanosta-7,24-dien-3β-ol。此外，在Tyr710Arg的突變株中也產生了兩個單環的產物， achilleol A和camelliol C。而所有突變點中只有Tyr710Pro使氧化鯊烯環化酵素失去活性，且沒有任何產物產生。由這些實驗結果，我們推測Tyr710可能透過與Trp390作用來影響單環C-10碳陽離子中間物的穩定性，並且在維持蛋白質的結構及活性區的形狀上有所貢獻。

The enzymatic cyclization of acyclic 2,3-oxidosqualene to polycyclic triterpenoids is one of the most complicated biochemical reactions that captured the interest of organic chemists and biochemists for over half a century. In fungi and mammals, oxidosqualene-lanosterol cyclase (ERG7) converts (3S)-2,3-oxidosqualene to the tetracyclic sterol precursor lanosterol via the so called “chair-boat-chair” conformation. The postulated cyclization/rearrangement reaction encompasses oxirane ring protonation and cleavage, cationic/π interaction-directed consecutive tetracyclic ring cyclization, 1,2-shifted hydride and methyl groups rearrangement, and final highly specific deprotonation step. Site-saturated mutagenesis experiments were carried out on two highly conserved aromatic residues, Tyr707 and Tyr710, of Saccharomyces cerevisiae ERG7 to investigate their functional roles in the oxidosqualene cyclization/rearrangement reaction. A novel bicyclic intermediate, (9R,10S)-polypoda-8(26),13E,17E,21-tetraen-3β-ol, with several altered deprotonation products were isolated from the ERG7Y707X mutants. It indicates that the Tyr707 residue may play an important role in stabilizing both the bicyclic C-8 cation and the final lanosteryl C-8/C-9 cationic intermediate. On the other hand, the site-saturated mutagenesis of ERG7Y710X revealed that two altered deprotonation products, parkeol and 9β-lanosta-7,24-dien-3β-ol as well as lanosterol, were produced in several mutants. Besides, the Tyr710Arg mutant produced two monocyclic products, achilleol A and camelliol C, and only the Tyr710Pro mutant lost the cyclase activity. These results suggested that Tyr710 may influence the stability of the monocyclic C-10 cationic intermediate through the interaction with Trp390 and play a role in preserving the cyclase structure and the conformation of active site.