Abstract
Protostane triterpenes, which are found in Alisma orientale, are tetracyclic triterpenes with distinctive pharmacological activities. The natural distribution of protostane triterpenes is limited mainly to members of the botanical family Alismataceae. Squalene epoxidase (SE) is the key rate-limiting enzyme in triterpene biosynthesis. In this study, we report the characterization of two SEs from A. orientale. AoSE1 and AoSE2 were expressed as fusion proteins in E. coli, and the purified proteins were used in functional research. In vitro enzyme assays showed that AoSE1 and AoSE2 catalyze the formation of oxidosqualene from squalene. Immunoassays revealed that the tubers contain the highest levels of AoSE1 and AoSE2. After MeJA induction, which is the main elicitor of triterpene biosynthesis, the contents of 2,3-oxidosqualene and alisol B 23-acetate increased by 1.96- and 2.53-fold, respectively. In addition, the expression of both AoSE proteins was significantly increased at four days after MeJA treatment. The contents of 2,3-oxidosqualene and alisol B 23-acetate were also positively correlated with AoSEs expression at different times after MeJA treatment. These results suggest that AoSE1 and AoSE2 are the key regulatory points in protostane triterpenes biosynthesis, and that MeJA regulates the biosynthesis of these compounds by increasing the expression of AoSE1 and AoSE2.
Highlights
Protostane triterpenes, which are found in Alisma orientale, are tetracyclic triterpenes with distinctive pharmacological activities
Protostane triterpenes are natural plant products with distinct pharmacological activities, but their natural distribution is limited to a few plant families[33,34]
The predicted protein sequences of AoSE1 and AoSE2 from A. orientale differ from other plant Squalene epoxidase (SE) protein sequences, but the functional domains are similar to those found in other SEs
Summary
Protostane triterpenes, which are found in Alisma orientale, are tetracyclic triterpenes with distinctive pharmacological activities. The contents of 2,3-oxidosqualene and alisol B 23-acetate were positively correlated with AoSEs expression at different times after MeJA treatment These results suggest that AoSE1 and AoSE2 are the key regulatory points in protostane triterpenes biosynthesis, and that MeJA regulates the biosynthesis of these compounds by increasing the expression of AoSE1 and AoSE2. Recent studies have shown that alisol B 23-acetate from A. orientale can restore the sensitivity of multi-drug resistant cells to anti-tumor drugs, because it may take part in the transport of P-glycoprotein[11] These compounds are found only in a few plant groups such as Alisma, and in very low amounts. SE catalyzes the conversion of squalene to 2,3-oxidosqualene, which is the precursor of the triterpene skeleton This enzyme is a non-cytochrome P450-type monooxygenase that participates in triterpene biosynthesis and functions as a rate-limiting step in the pathway[18]. In previous studies on the key enzymes in the protostane triterpenes biosynthetic pathway in A. orientale, we cloned and characterized AoHMGR, AoFPPS, and AoSS (GenBank accession numbers KP342318, HQ724508, and JX866770, respectively)[23,24,25], while characterization and functional analysis of SE in A. orientale has not yet been reported
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