罗汉果环阿屯醇合酶的同源建模、分子对接及催化环化的机理推测  

Homology Modeling and Molecular Docking of Cycloartenol Synthase in Siraitia grosvenorii and Speculated Mechanism of Catalytic Cyclization

作　　者：乔晶 崔晟榕 石宏武[1] 罗祖良[1] 马小军[1] QIAO Jing;CUI Sheng-rong;SHI Hong-wu;LUO Zu-liang;MA Xiao-jun(Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences & Peking Union Medical College,Beijing 100193)

机构地区：[1]中国医学科学院北京协和医学院药用植物研究所,北京100193

出　　处：《生物技术通报》2019年第2期101-108,共8页Biotechnology Bulletin

基　　金：国家自然科学基金资助项目(81573521);北京市自然基金资助项目(5172028)

摘　　要：环阿屯醇为植物甾醇类化合物,也是诸多甾醇类化合物生物合成的关键前体物质之一,具有抗炎、抗氧化、抗肿瘤、调节胆固醇等多种药理活性。课题组前期克隆了罗汉果环阿屯醇合酶基因(Cycloartenol Synthase,CAS)并对其进行了功能验证(GenBank登录号:HQ128566)。但该酶的催化活性位点及催化机制尚不明确,阻碍了进一步的改造及应用。本研究利用同源建模预测CAS的三维结构,结合分子对接模拟技术分析该酶与底物的相互作用,结果表明,Asp491、Cys492、Cys570、Tyr540、His265是CAS酶上关键的催化位点,Asp491质子化2,3-氧化鲨烯引发四个环化反应形成C20正离子中间体,然后中间体经过一系列的碳正离子重排形成C11正离子中间体,最后通过His265与Tyr540去质子化使得C27与C11原子间形成单键生成产物环阿屯醇。此外,活性空腔内存在大量的疏水氨基酸,则通过疏水作用稳定反应物、中间体结合在活性空腔。该酶活性位点的发现,为今后通过定点突变技术改造酶的活性及调控代谢通路奠定了基础。Cycloartenol,a phytosterol compound,also one of the key precursor substances for biosynthesis of numerous sterol compounds,demonstrates a variety of pharmacological activities such as anti-inflammatory,antioxidant,anti-tumor and regulating cholesterol levels.The genes of cycloartenol synthase(CAS)from Siraitia grosvenorii cloned in our team’s previous work have been functionally characterized(GenBank accession number:HQ128566).However,its active sites and catalytic mechanism of cyclization remain unclear,which hinders its modification and application.Homology modeling was used to predict the 3D structure of CAS,and the interaction between CAS and substrates was analyzed by combining with molecular docking.Results showed that Asp491,Cys492,Cys570,Tyr540,and His265 were the key catalytic sites in CAS.The catalytic mechanism for the cyclization reaction were involved several reaction steps.First,4 cyclization reactions were triggered by Asp491 protonating 2,3-oxidosqualene and C20 cation intermediate was formed.Then,skeletal rearrangement of C cation in this intermediate resulted in the formation of C11 intermediate.Finally,deprotonating by His26 and Tyr540 led to the product cycloartenol via forming single bond between C27 and C11.In addition,there were a large number of hydrophobic amino acids in the active cavity,and the product was stabilized by hydrophobic reaction and the intermediate bound to the active cavity through the hydrophobic action.The discovery of enzyme active sites laid a foundation for the modification of enzyme activity by site-directed mutation technique and regulation of metabolic pathway in the future.

关 键 词：环阿屯醇合酶 同源建模 分子对接 活性位点 催化机制 

分 类 号：Q55[生物学—生物化学]