Exploiting alkaloid biosynthesis in Madagascar periwinkle to obtain natural product derivatives and new biocatalysts

Abstract

Plant alkaloid biosynthesis produces many natural products with medicinal value. For example, vinblastine and vincristine from Catharanthus roseus monoterpene indole alkaloid biosynthesis, and camptothecin derivatives from Ophiorrhiza pumila quinoline alkaloid biosynthesis, are anticancer agents currently used in the clinic. Strictosidine synthase is a key enzyme in the biosynthesis of these medicinal natural products, but its narrow substrate scope limits precursor-directed biosynthesis of alkaloid analogs in plant cell cultures. I describe two new assays to monitor strictosidine synthase activity, which enable the rapid screening of enzyme mutant libraries to identify two strictosidine synthase variants that accept new substrates. A transgenic plant cell culture that contains one of these mutants generated "unnatural" monoterpene indole alkaloids in C. roseus. I also describe the characterization of 0. pumila strictosidine synthase, which has considerably broader substrate specificity than the homologous enzyme from C. roseus. This alternative catalyst is a candidate enzyme for construction of transgenic cell cultures, and potentially useful as a biocatalyst, since it catalyzes the asymmetric Pictet- Spengler reaction to form tetrahydro-p-carboline pharmacophores. I used computer modeling to propose a model for how strictosidine synthase achieves its high stereoselectivity; this model may be used to engineer a Pictet-Spenglerase that forms the alternative stereoisomer.

(cont.) Such a stereocomplementary catalyst would be useful in biocatalysis, giving the synthetic organic chemist access to both stereoisomers in high enantiomeric purity. Finally, I describe the total synthesis of stereoisomer mixtures of indole alkaloid precursors, and use these mixtures to determine the stereoselectivity of strictosidine synthase and two subsequent enzymes in monoterpene indole alkaloid biosynthesis. The combination of chemical synthesis and the recruitment of enzymes from unrelated biosynthetic pathways could generate diverse alkaloid libraries, containing different stereoisomers, for bioactivity evaluation.