Abstract
Triterpenoids exist in a free state and/or in conjugated states, such as triterpene glycosides (saponins) or triterpene esters. There is no information on the enzyme participating in the production of triterpene esters from free triterpenes. Lettuce (Lactuca sativa) contains various pentacyclic triterpene acetates (taraxasterol acetates, ψ-taraxasterol acetates, taraxerol acetates, lupeol acetates, α-amyrin acetates, β-amyrin acetates, and germanicol acetate). In this study, we report a novel triterpene acetyltransferase (LsTAT1) in lettuce involved in the biosynthesis of pentacyclic triterpene acetates from free triterpenes. The deduced amino acid sequences of LsTAT1 showed a phylogenetic relationship (43% identity) with those of sterol O-acyltransferase (AtSAT1) of Arabidopsis thaliana and had catalytic amino acid residues (Asn and His) that are typically conserved in membrane-bound O-acyltransferase (MBOAT) family proteins. An analysis of LsTAT1 enzyme activity in a cell-free system revealed that the enzyme exhibited activity for the acetylation of taraxasterol, ψ-taraxasterol, β-amyrin, α-amyrin, lupeol, and taraxerol using acetyl-CoA as an acyl donor but no activity for triterpene acylation using a fatty acyl donor. Lettuce oxidosqualene cyclase (LsOSC1) is a triterpene synthase that produces ψ-taraxasterol, taraxasterol, β-amyrin and α-amyrin. The ectopic expression of both the LsOSC1 and LsTAT1 genes in yeast and tobacco could produce taraxasterol acetate, ψ-taraxasterol acetate, β-amyrin acetate, and α-amyrin acetate. However, expression of the LsTAT1 gene in tobacco was unable to induce the conversion of intrinsic sterols (campesterol, stigmasterol, and β-sitosterol) to sterol acetates. The results demonstrate that the LsTAT1 enzyme is a new class of acetyltransferase belong to the MBOAT family that have a particular role in the acetylation of pentacyclic triterpenes and are thus functionally different from sterol acyltransferase conjugating fatty acyl esters.
Highlights
Triterpenoids are diverse and abundant natural products that consist of six isoprene units, are categorized according to the structures of their triterpene rings and exhibit various biological and pharmacological activities (González-Coloma et al, 2011; Yang et al, 2020)
Among the 12 triterpenes, six compounds were detected as triterpene esters (α-amyrin acetate, β-amyrin acetate, lupeol acetate, ψ-taraxasterol acetate, taraxasterol acetate, and germanicol acetate) as major constituents in the leaves of all four cultivars (Figures 1A,B)
All six triterpene esters were esterified by acetic acid, as identified by a comparison with standard triterpene acetate compounds (Figure 1)
Summary
Triterpenoids are diverse and abundant natural products that consist of six isoprene units, are categorized according to the structures of their triterpene rings and exhibit various biological and pharmacological activities (González-Coloma et al, 2011; Yang et al, 2020). Triterpenoids exist in a free state and in conjugated states, such as triterpene glycosides (saponins) or triterpene esters (Thimmappa et al, 2014). Triterpene glycosylation or acylation is an important phenomenon for diversifying the structure of triterpenes and may affect biological function (Osbourn et al, 2011). Acylated triterpenes esterified by acetic acid and fatty acids are very common in many plant species (Warnaar, 1987; Catharina et al, 2013; Hill and Connolly, 2017; Choi et al, 2020). Triterpene acetates are found in the soil of plant vegetation and can be used as potential chemotaxonomic markers of Asteraceae to track past vegetation changes (Lavrieux et al, 2011; Nakamura, 2019)
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