Cloning and characterization of triterpene synthases

Oxidosqualene cyclases are a group of enzymes that cyclize 2,3-oxidosqualene to any of over 80 triterpene skeletons. Sequence correlation suggests these enzymes share a common overall structure and subtle changes in active site residues are responsible for differences in the structure of the cyclization product. Further characterization of conserved residues has lead to a better understanding of the mechanisms through which regio- and stereo-selective cyclization occurs. This work has resulted in the cloning of three new oxidosqualene cyclases. By cloning triterpene synthases from far-diverged organisms, new sequence data has been added for correlation and has provided new information regarding evolution of the enzymes from a common ancestor. Arabidopsis thaliana lupeol synthase was cloned and the full-length cDNA expressed in yeast. Degenerate PCR primers designed from regions of highly conserved sequence were used to clone cycloartenol synthase and a presently uncharacterized triterpene synthase from an Abies magnifica library. Newly obtained and previously published sequence data has been used to identify residues that may correlate with catalytic activity. Site-directed mutagenesis has been performed on Arabidopsis thaliana cycloartenol synthase resulting in enzymes with different product distributions. In addition to producing cycloartenol, these mutant proteins were shown to produce a range of new triterpene alcohols, including monocyclic alcohols and a previously unknown triterpene skeleton. Sequence information as well as the mechanistic information learned from the analysis of mutant protein products has come together to reveal a complex relationship between structure and function of triterpene synthases. Using techniques of molecular biology as well as analytical and organic chemistry, aspects of the nature of oxidosqualene cyclases in terms of the mechanisms of transformation, evolution and physiological significance are presented