Diterpenoid metabolism in Mycobacterium tuberculosis

Given our interest in labdane-related diterpenoid biosynthesis, we were intrigued by the report that the important human pathogen Mycobacterium tuberculosis encoded a class II diterpene cyclase, particularly with separately reported genetic evidence that this enzyme was involved in the ability of the pathogen to suppress acidification of the phagosome into which the bacterium is enveloped by macrophages.

Diterpenoids in plant-microbe interactions

The observed diversification of (di)terpenoids in plants suggests that these natural products play important roles in such organisms.  Chief among these are the gibberellin phytohormones, produced by both plants and associated microbes, and which have given rise to the particularly extensive super-family of labdane-related diterpenoids.  To demonstrate the physiological relevance of these natural products, we have been investigating their role in plant-microbe interactions.  Based on our extensive work on rice labdane-related diterpenoid metabolism, we have taken a reverse genetic approach to elucidate their role in the plant.

Metabolic Engineering

Given our broad interest in (di)terpenoid metabolism, we developed a modular metabolic engineering system that enables facile assembly of diterpenoid biosynthetic pathways in E. coli.  While we have carried out some further engineering to increase flux towards terpenoid metabolism, this is not a primary focus.  In particular, we are not interested in pursuing large-scale production of any given compound, but rather developed our system as a platform for discovery and exploration of relevant biosynthetic enzymes.

Mechanistic studies of class II diterpene cyclases

The class II diterpene cyclases catalyze protonation-initiated (bi)cyclization reactions that characterize labdane-related diterpenoid biosynthesis. In collaboration with Prof. David Christianson (Univ. Penn), we have provided the first crystal structures for class II diterpene cyclases.  From these we have generated significant insights into the underlying determinants of enzymatic catalysis, including the generation of enzymes wherein single residue changes have led to novel product outcome.

Mechanistic studies of class I diterpene synthases

Recognizing that rice produced numerous labdane-related diterpenoid natural products, and the opportunity presented by the publication of the rice genome sequence, we began by carrying out a functional genomics based investigation of the rice diterpene synthases.  Given the relative paucity of characterized diterpene synthases at that time, this effort more than doubled the number of molecularly identified functionally distinct such enzymes.  Strikingly, we further uncovered a functionally distinct pair of alleles for one of the rice class I diterpene synthases, which led to isolation of a single residue ‘switch’ for product outcome.