Abstract
The terpenoid family represents one of the most abundant and diverse classes of organic molecules found in nature. Because limonene synthase catalyses the simplest of all monoterpene (C10) cyclization reactions, it is often used as a model for monoterpene biosynthesis. The study described here was done with the enzyme (+)-(4R)-limonene synthase, which was cloned previously from the flavedo of Citrus sinensis. In this thesis, we discuss the kinetic characterization of this enzyme, in which we observe kinetic properties very different from those previously found for the same enzyme cloned from Citrus limon. We also describe a crystal structure of this enzyme in apoprotein form, and the soaking of such crystals in a substrate-containing solution, where we observe time-dependent changes in electron density for three coordinating Mn2+ atoms in the enzyme active site. We hope that these results constitute key steps in identifying the structural components of the enzyme mechanism. Finally, we relate the initial stages of an effort to produce (+)-limonene, a potential biofuel, in the cyanobacterium Synechocystis by engineering our (+)-(4R)-limonene synthase gene into the Synechocystis genome.