Catalytic and regulatory mechanisms of diterpene synthesis in plants and bacteria.

摘要

Labdane-related diterpenoids are an important family of natural products with over 7,000 known members and new structures continually being characterized. These important biomolecules serve vital roles in signaling and defense in their host systems, and several also have potent pharmaceutical properties. Elucidating the biosynthetic mechanisms of diterpenoid metabolism is key to understanding and harnessing their biological potential. Though the regulation of diterpenoid product biosynthesis has been extensively studied in plants via metabolite profiling and transcriptional studies, catalytic mechanisms and biochemical regulation require clarification. This regulation occurs in the committed step of diterpenoid biosynthesis: the class II diterpene synthase. In this thesis, I present such analylsis of the catalytic mechanism as well as mechanistic evidence for divalent cation dependent regulation of two diterpenoid biosynthetic pathways: the Gibberellic acid biosynthesis pathway in higher plants and a related pathway in Mycobacterium tuberculosis, which is likely involved in biosynthesis of the immune suppressant metabolite, isotuberculosinol. Through the analysis of catalytic residues and kinetic properties of the plant enzyme, we are able to better understand the regulatory mechanisms of diterpenoid biosynthesis in M. tuberculosis. In addition, the characterization of isotuberculosinol, as well as genetic characterization of the related biosynthetic pathway, lends insight into the pathogenic mechanisms of M. tuberculosis. With this new information, a variety of future experiments to test, confirm, or elaborate on current hypotheses, are proposed. This work represents a varied illustration of how understanding the mechanistic aspects of diterpenoid metabolism can lend insight into metabolic processes within the whole organism and, more broadly, metabolic evolution.