Dissertation Defence: Biosynthesis of Spirooxindole Alkaloids

Minh Tuan Anh Nguyen, supervised by Dr. Thuy Dang, will defend their dissertation titled “Biosynthesis of Spirooxindole Alkaloids” in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry.

An abstract for Minh Tuan Anh Nguyen’s dissertation is included below.

Examinations are open to all members of the campus community as well as the general public. Registration is not required for in-person exams.

Abstract

Monoterpene indole alkaloids (MIAs) constitute one of the largest and most structurally diverse classes of plant specialized metabolites with significant pharmaceutical importance. Spirooxindoles, a subclass of MIAs, contain a unique three-dimensional spiro[pyrrolidine-3,3-oxindole] ring system recognized as a valuable pharmacophore in drug discovery. However, their biosynthesis in plants has remained elusive. This conformationally constrained scaffold is found in natural products exhibiting anticancer, anti-inflammatory, antimicrobial, and other pharmacological activities. Its structural complexity, however, also makes spirooxindoles challenging targets for chemical synthesis, emphasizing the importance of identifying the enzyme that construct this scaffold in plants. This dissertation addressed this knowledge gap through gene discovery, pathway reconstruction, and mechanistic studies. Multi-omics analyses in the Mitragyna genus enabled the first identification and functional characterization of the previously unknown enzyme system underlying spirooxindole biosynthesis. This system comprises a flavindependent oxidase (FAD), a short-chain reductase (SDR), and a cytochrome P450 (P450/CYP). Biochemical and heterologous pathway-reconstitution experiments demonstrated that the flavoprotein and reductase act sequentially as stereochemical “gatekeepers”, converting (3S)-corynanthe alkaloids into their (3R) epimers required by the downstream P450, which cannot directly accept the naturally abundant (3S) substrates. The newly identified cytochrome P450, designated spirooxindole synthase (SOS), solely accepts the (3R)-corynanthe substrates and catalyzes their oxidative rearrangement into the corresponding spirooxindoles, representing the first reported plant enzyme to construct a spirooxindole scaffold and revealing a plant-specific, P450-dependent solution to this chemically demanding skeletal rearrangement. To study the iv mechanism of spirooxindole formation, the plant membrane-bound SOS was expressed and purified in Escherichia coli using a newly established, robust, and reproducible method. Protein modelling with AlphaFold and site-directed mutagenesis revealed the core residues involved in substrate binding and catalysis. Mechanistic studies using isotopic labelling further supported an initial substrate epoxidation and a semi-pinacol-type rearrangement to yield the defining spirooxindole centre. These discoveries revealed the three-enzyme system governing stereochemical inversion and spirooxindole scaffold formation in plants, thereby establishing a previously unrecognized strategy for MIA diversification. The identified oxidase–reductase–P450 module also provides a foundation for reconstructing spirooxindole pathways in engineered hosts and for developing biocatalytic routes to pharmaceutically relevant natural products and new-tonature analogues.