The enzymatic conversion of oxidosqualene to cyclic triterpenes represents the key step in the biosynthesis of more than 20,000 triterpenoids. These compounds are widely distributed in nature among plants, animals, fungi, and some bacteria. Triterpenes serve as precursors of sterols, steroids, and numerous secondary metabolites of undefined function. Insights into triterpene biochemistry are emerging from studies of gene expression, metabolite profiles, and enzyme function in the reference plant Arabidopsis thaliana . This thesis describes the functional characterization by heterologous expression in Saccharomyces cerevisiae of four Arabidopsis oxidosqualene cyclases: PEN3 (At5g36150), LUP1 (At1g78970), LUP2 (At 1g78960), and LUP5 (At1g66960). PEN3, the last uncharacterized Arabidopsis cyclase makes predominantly tirucalla-7,24-dienol. The elucidation of the product profile of PEN3 illuminated alternative mechanistic routes to the formation of tetracyclic triterpene alcohols by dammarenyl-type oxidosqualene cyclases. LUP5, which had only been cursorily characterized, makes the same set of 6/6/6/5 tetracycles as that of PEN3. This represents a likely example of convergent evolution since these genes show rather low sequence identity. I found that LUP1 is a lupane-3β,20-diol (lupanediol) synthase not a lupeol synthase or a multifunctional enzyme. The fact that lupanediol is the major LUP1 product suggests that lupanediol is the biosynthetic precursor of trinorlupeol, a major nonsterol triterpenoid in Arabidopsis . The abundance of minor LUP1 products may reflect the difficulty for cyclases to make diols accurately. LUP2 is a truly multifunctional enzyme, making a mixture of β-amyrin and other cyclic triterpenes, any of which could have been better optimized by selective pressure to make a single product. I established that the product profile of LUP2 and LUP1 in previous characterizations was distorted by selective loss of triterpene products to the culture medium. These results underlined the importance of analyzing the medium to avoid misleading interpretation of in vivo product profiles. The characterization of PEN3 completes the first functional characterization of all triterpene synthases in a higher plant. These results together with more reliable product profiles of some LUP genes provide a better understanding of the catalytic properties of this class of enzymes.
Morlacchi, Pietro. "Triterpene biosynthesis in plants: Oxidosqualene cyclization in Arabidopsis thaliana." (2009) Diss., Rice University. https://hdl.handle.net/1911/103744.