Biochemistry and Molecular Biology of Sesquiterpene Cyclase and Squalene Synthetase from Tobacco

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9408152 Chappell Isoprenoids are ubiquitous in nature, essential for life and mediate a number of interactions between organisms and their environment. In addition, plants are a rich source of isoprenoids important for pharmaceutical uses. Yet, our understanding of the isoprenoid biosynthetic pathway in plants is limited. Recent studies indicate that tobacco cell suspension cultures are uniquely suited for detailed physiological and biochemical studies of one important but putative branch point in isoprenoid metabolism. When fungal elicitors are added to the cell cultures, the cultures cease sterol production and instead synthesize and secrete anti-microbial sesquiterpenoids. The decline in sterol biosynthesis has been correlated with suppression of squalene synthetase enzyme activity, and the induction of sesquiterpenoid biosynthesis with the induction of a sesquiterpene cyclase. Because these two enzymes are positioned at a putative branch point in the pathway, the induction of one enzyme and the suppression of the other are interpreted as an important regulatory mechanism controlling carbon flow and hence, end product formation. Alternatively, independently regulated arrays of isozymes dedicated to the production of specific end products might be aligned along a surface or membrane. Regardless of which metabolic organization might be occurring within a plant cell, squalene synthetase and sesquiterpene cyclase are key enzymes for sterol and sesquiterpene biosynthesis. The long range goal of this research is to understand all aspects of these two enzymes, from a complete understanding of their enzymology to the fine details of how expression of the corresponding genes are regulated. The current objectives are to continue defining the DNA sequences and DNA binding proteins important for controlling the transcription rate of the cyclase and squalene synthetase genes, and to begin an analysis of the cyclase reaction mechanism using a combination of comparative molecular biology and an evaluation of chimeric cyclase genes expressed in bacteria. Results from these studies will help elucidate the mechanisms regulating isoprenoid metabolism in plants, and will have important ramifications for future manipulations of this pathway using genetic engineering technology. ***