标题: 利用饱和定点突变方法探讨酵母菌氧化鲨烯环化酵素其胺基酸Val454与Asn700的功能并搭配表现之阿拉伯芥转醣酵素进行固醇化合物转醣衍生化的研究
Functional Analysis of Val454 and Asn700 Site-Saturated Mutants within Saccharomyces cerevisiae Oxidosqualene Lanosterol Cyclase and Using Expressed Arabidopsis thaliana Sterol Glucosyltransferases for Sterols Derivatization
作者: 宋欣怡
吴东昆
生物科技学系
关键字: 氧化鲨烯环化酵素;阿拉伯芥转醣酵素;固醇化合物转醣;Oxidosqualene Lanosterol Cyclase;Arabidopsis thaliana;Sterol Glucosyltransferases
公开日期: 2010
摘要: 氧化鲨烯环化酵素在真菌、哺乳类动物或者高等植物中催化氧化鲨烯进行环化和重组反应。此催化反应透过环氧基开环、多烯与碳阳离子间之诱导环化、氢化基和甲基之重组反应及最后具有高度专一性的去质子步骤,使得直链状之氧化鲨烯环化形成多环的三萜类产物。在氧化鲨烯环化酵素的酵素活性区域内,有多个位在第一层或第二层的胺基酸已被证实,其对于稳定碳阳离子中间物或酵素催化活性具有很重要的关系。为了更进一步阐明其他胺基酸在氧化鲨烯环化酵素内所扮演的角色,我们利用酵母菌的氧化鲨烯环化酵素,针对其缬胺酸454号胺基酸 (Val-454) 和天冬醯胺酸700号胺基酸 (Asn-700) 进行饱和定点突变实验,以探讨此两个胺基酸残基在氧化鲨烯环化酵素中其功能性之角色。根据所进行的饱和突变产物分析结果,我们对缬胺酸454号胺基酸和天冬醯胺酸700号胺基酸的催化角色已有所了解。天冬醯胺酸700号胺基酸可能经由突变作用直接影响涵盖苯丙胺酸699号胺基酸 (Phe-699)、异白胺酸705号胺基酸 (Ile-705) 至酪胺酸707号胺基酸 (Tyr-707) 活性区段的催化能力,因此天冬醯胺酸700号胺基酸对于多个碳阳离子中间产物具有重要的稳定作用;缬胺酸454号胺基酸其侧链可能是辅助B环环化与帮助去质子步骤的一个重要角色,突变产物结果也指出,缬胺酸454号胺基酸可以稳定碳-10中间产物。这些研究证据均表示缬胺酸454号胺基酸和天冬醯胺酸700胺基酸位置对于氧化鲨烯环化酵素其一连串的催化作用,具有举足轻重的角色。此外由于氧化鲨烯环化酵素的环化反应已被本实验室研究多年,大致的环化机制已充分地被了解,所以我们想利用突变后的产物来开辟一个新的研究方向。因此我们转殖、表现与纯化阿拉伯芥中的一个转醣酵素,并在胶凝体电泳上可看到一个约70千道尔吞胺基酸大小的蛋白质。此外我们利用基质辅助雷射吸附质谱技术成功鉴定出此纯化蛋白为阿拉伯芥转醣酵素,且利用偶合酵素法确认其对于胆固醇分子具有转糖活性。由实验结果显示,其能催化四环固醇类产物转变成固醇配醣体,我们期望利用此转醣酵素针对我们所获得的多个突变株其三萜类产物进行衍生化,以祈能利用这些衍生化产物应用于未来药物学方面的研究。
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.
URI: http://140.113.39.130/cdrfb3/record/nctu/#GT079828536
http://hdl.handle.net/11536/47735
显示于类别:Thesis