Fig. 4: Functional characterisation of a pot marigold Ψ-taraxasterol synthase (Co TXSS).
From: Biosynthesis and bioactivity of anti-inflammatory triterpenoids in Calendula officinalis
A Biosynthesis of Ψ-taraxasterol, taraxasterol, α-amyrin, β-amyrin and lupeol by 2,3-oxidosqualene cyclases. B Structural models showing the predicted position of the taraxasteryl cation in the active site of CoTXSS and CoMAS (mixed amyrin synthase). Residues that differ are highlighted in blue. C Phylogenetic tree and sequence alignment of selected residues of MASs and TXSSs with active site residues highlighted in grey and residues selected for mutagenesis in dark grey. Ia Ilex asprella; Cr Catharanthus roseus; Oe Olea europaea; Ob Ocimum basilicum; Co Calendula officinalis; Aa Artemisia annua; Ha Helianthus annuus; Cc Cynara cardunculus; Td Tragopogon dubius; Ls Lactuca sativa; Ce Cichorium endivia; Tk Taraxacum kok-saghyz; Tc Taraxacum coreanum. A list of taxa and accession numbers/protein sequences are provided in Supplementary Table 4. D Total ion chromatograms of extracts of N. benthamiana leaves transiently expressing wild type and mutated CoMAS and CoTXSS. E Quantification of triterpenes produced by wild type and mutated CoMAS and CoTXSS. Error bars indicate the mean and standard error of 6 biological replicates (independent infiltrations). Significant differences in α/β-amyrin content (blue lowercase letters) and Ψ-taraxasterol/taraxasterol content (red lowercase letters) were analysed using a Kruskal-Wallis test followed by post-hoc Wilcoxon rank sum test with a Benjamini-Hochberg correction (Supplementary Data 7). Samples that do not share the same lower-case letter are significantly different from each other (p < 0.05). P values are provided in Supplementary Data 7.
