利用飽和定點突變方法探討酵母菌氧化鯊烯環化酵素其胺基酸Val454與Asn700的功能並搭配表現之阿拉伯芥轉醣酵素進行固醇化合物轉醣衍生化的研究

Abstract

氧化鯊烯環化酵素在真菌、哺乳類動物或者高等植物中催化氧化鯊烯進行環化和重組反應。此催化反應透過環氧基開環、多烯與碳陽離子間之誘導環化、氫化基和甲基之重組反應及最後具有高度專一性的去質子步驟，使得直鏈狀之氧化鯊烯環化形成多環的三萜類產物。在氧化鯊烯環化酵素的酵素活性區域內，有多個位在第一層或第二層的胺基酸已被證實，其對於穩定碳陽離子中間物或酵素催化活性具有很重要的關係。為了更進一步闡明其他胺基酸在氧化鯊烯環化酵素內所扮演的角色，我們利用酵母菌的氧化鯊烯環化酵素，針對其纈胺酸454號胺基酸 (Val-454) 和天冬醯胺酸700號胺基酸 (Asn-700) 進行飽和定點突變實驗，以探討此兩個胺基酸殘基在氧化鯊烯環化酵素中其功能性之角色。根據所進行的飽和突變產物分析結果，我們對纈胺酸454號胺基酸和天冬醯胺酸700號胺基酸的催化角色已有所了解。天冬醯胺酸700號胺基酸可能經由突變作用直接影響涵蓋苯丙胺酸699號胺基酸 (Phe-699)、異白胺酸705號胺基酸 (Ile-705) 至酪胺酸707號胺基酸 (Tyr-707) 活性區段的催化能力，因此天冬醯胺酸700號胺基酸對於多個碳陽離子中間產物具有重要的穩定作用；纈胺酸454號胺基酸其側鏈可能是輔助B環環化與幫助去質子步驟的一個重要角色，突變產物結果也指出，纈胺酸454號胺基酸可以穩定碳-10中間產物。這些研究證據均表示纈胺酸454號胺基酸和天冬醯胺酸700胺基酸位置對於氧化鯊烯環化酵素其一連串的催化作用，具有舉足輕重的角色。此外由於氧化鯊烯環化酵素的環化反應已被本實驗室研究多年，大致的環化機制已充分地被了解，所以我們想利用突變後的產物來開闢一個新的研究方向。因此我們轉殖、表現與純化阿拉伯芥中的一個轉醣酵素，並在膠凝體電泳上可看到一個約70千道爾吞胺基酸大小的蛋白質。此外我們利用基質輔助雷射吸附質譜技術成功鑑定出此純化蛋白為阿拉伯芥轉醣酵素，且利用偶合酵素法確認其對於膽固醇分子具有轉糖活性。由實驗結果顯示，其能催化四環固醇類產物轉變成固醇配醣體，我們期望利用此轉醣酵素針對我們所獲得的多個突變株其三萜類產物進行衍生化，以祈能利用這些衍生化產物應用於未來藥物學方面的研究。

Oxidosqualene cyclases (OSCs) catalyze the cyclization/rearrangement of (3S)-2,3-oxidosqualene into the polycyclic triterpenoids in fungi, mammals, or high plants through a protonated oxirane ring cleavage coupled with the formation of a conformationally prefolded cationic intermediate, followed by four or five cationic- olefinic-mediated ring annulations, a series of 1,2-shifted hydrides or methyl groups’ rearrangement, and a highly specific deprotonation, respectively. Various residues in either first- or second-tier active sites of OSCs have showed their own importance in stabilizing the respective carbocationic intermediates and enzyme catalysis. In order to further clarify the roles of other amino acids in oxidosqualene-lanosterol cyclase, site-saturated mutagenesis of ERG7N700X and ERG7V454X from Saccharomyces cerevisiae were carried out. From the results of the genetic selection and product profiles characterization of these site-specific mutants, the catalytic roles of these two highly conserved residues have thus been unraveled. Asn700 showed its functional importance in stabilizing various carbonic intermediates via its mutagenic effects on an enzyme active site region which contains three critical amino acids, Phe699, Ile705, and Tyr707. The Val454 side chain may participate in the stabilization of the monocyclic C-10 cationic intermediate and facilitate the B-ring formation as well as the deprotonation step, indicated by the analysis of saturated-mutants product profiles. These experimental evidences represented the roles of ERG7N700X and ERG7V454X involved in the OSC-catalyzed reaction cascades. In parallel, the molecular cloning, over-expression, chromatographic purification, and biophysical characterization of one sterol glucosyltransferases (AtSGT) from Arabidopsis thaliana have been carried out. The purified protein about 70 kDa molecular mass was revealed on SDS-PAGE and the trypsin digested fragments and one single charged precursor m/e at 1947.693 were resolved by MALDI-TOF/MS mass spectrometry, that identify the purified protein as AtSGT. AtSGT was showed to catalyze the glycosylation of cholesterol by couple enzyme assay by using the UDP-activated sugar as a glycosyl group donor. AtSGT will be used to derivatize various truncated cycliczation/deprotonation triterpene products isolated from OSC mutants. Those structurally derivative analogs will open the avenue for applying these OSC mutants-generated products in pharmaceutics.