Abstract
Scientists have long marveled at the structural diversity of terpenoids and have speculated on its biosynthetic basis. Limonene synthase is often considered a model enzyme for terpenoid formation since it catalyzes the mechanistically simplest cyclization reaction. In this study, we attempt to address the question of how this class of enzymes achieves its regio- and stereospecificity, by supplementing previous studies on (-)-4S-limonene synthase with studies on (+)-4R-limonene synthase. Comparative structure and function analyses of these two enzymes, catalyzing the formation of the enantiomeric products (-)- limonene and (+)-limonene, respectively, will shed new light on differences in structural features and how these can direct stereoisomeric outcomes. Our study is the first to report successful isolation and functional expression of (+)-4R-limonene synthase from Citrus sinesis. We furthermore showed our clone to be enzymatically active, albeit with a higher Km than previously reported for other monoterpene synthases. Although various problems with our expression and purification protocol need to be addressed before crystallization trials can be set up, the isolation of an enzymatically active clone from orange tissue represents meaningful progress towards the ultimate goal of determining structural features that provide a mechanism for the skeletal diversity so characteristic of this class of enzymes.