Investigations into the structure-function relationship of the conserved aromatic residues in Class II Diterpene Cyclases

Abstract

Terpenoids comprise the largest and most chemically diverse class of naturally occurring organic products. Their ubiquitous presence in all kingdoms of life and remarkable scope of function have made their biosynthetic pathways of great interest in a plethora of commercial sectors. Terpenes are non-bioactive precursors of terpenoids and derive from 5-carbon isoprene subunits that are coupled through sequential condensation to form linear chains of varying lengths. Diterpenes form the 20-carbon subclass of terpenes and are destined for cyclization and functional decoration to produce bioactive diterpenoids. The labdane-related diterpenoids form a particularly important subclass of terpenoids since they have been associated with containing many industrially desired characteristics through their inherent anti-bacterial, anti-inflammatory, anti-fungal, and anti-tumor properties. The class II diterpene cyclases (DTCs) are the group of enzymes responsible for catalyzing the committed step in the biosynthesis of all labdane-related diterpenoids. This particular class of enzyme utilizes a general acid-base mediated cascade reaction for the bicyclization of the common diterpenoid precursor (E, E, E)-geranylgeranyl diphosphate (GGPP). In this mechanism, the substrate is first protonated to form a carbocation, which can then be subsequently deprotonated by various methods to form diverse cyclized products. Present within the active sites of DTCs are conserved motifs that are thought to contribute to the overall catalytic function of the enzyme. One such motif is comprised of the conserved aromatic residues, which are hypothesized to provide structural integrity to the active site, as well as stabilize the carbocation intermediate during catalysis. Investigations in this thesis employ mutagenesis and metabolic engineering studies to probe the structure-function relationship of the conserved aromatic residues in DTCs, particularly between the plant and bacterial kingdoms. The data presented here has provided insight into the functional effects of the conserved aromatic residues with regard to the alteration of both product outcome and yield, as well as contributed to the reservoir of knowledge concerning the evolutionary divergence of DTCs.