Characterization of Oxidosqualene Cyclases in Brassicaceae: the ABCs (Arabidopsis, Brassica and Capsella)
Matsuda, Seiichi P. T.
Doctor of Philosophy
One major class of plant secondary metabolites is the triterpenes, whose biosynthesis from oxidosqualene is catalyzed by oxidosqualene cyclases (OSCs). This thesis describes the characterization of OSCs from plants within the Brassicaceae family, which include the model species Arabidopsis thaliana and Capsella rubella as well as the crop species Brassica rapa and Brassica oleracea. Detailed product profiles of those cyclases are combined with phylogenetic analysis, synteny analysis, active-site prediction, plant tissue extraction and transcriptome analysis to explore enzyme mechanisms and evolutionary relationships within the OSC family. First, detailed product profiles are constructed for four Arabidopsis thaliana OSCs. Those cyclases exemplify the range of product specificity among OSCs, ranging from the 94% specific At PEN4 to the multi-functional cyclase At LUP5 which makes two major products at similar levels. Despite their varying product specificity, all those OSCs form many minor products. Next, the characterization of three OSCs from Brassica species is detailed. Bra032185 was determined to be an astertarone A synthase, which is the first reported OSC with 6/6/6/5 20R stereospecificity. Bra039929 is the first reported euphol synthase and is another 6/6/6/5 20R specific cyclase. In addition, Bol021540 was amplified from broccoli seedling RNAs and it was determined to be a mixed-amyrin synthase. Those results were combined with Brassica plant extraction results to explore the triterpene biosynthesis in this species. Lastly, the complete characterization of all Capsella rubella OSCs is described. The triterpene biosynthetic capability of this plant includes one cycloartenol synthase, five functional OSCs in the secondary metabolism and one pseudogene. The only secondary metabolic OSC conserved between A. thaliana and C. rubella is camelliol C synthase (LUP3). In addition, three C. rubella cyclases show similarity to their A. thaliana counterparts but different products are made. Overall, the comparison of OSCs across species illustrates the fast divergence of plant secondary metabolism and explores OSC evolution among the Brassicaceae family. The OSC family showcases rapid enzyme functional evolution as evident by neofunctionalization and convergent evolution, and thus it can be a valuable model for enzyme evolution studies.