Abstract: 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.

Key words: cycloartenol synthase, homology modeling, molecular docking, active sites, catalytic mechanism of cyclization