Estimates suggest the plant kingdom possesses the capacity to synthesize up to 1,000,000 metabolites. While a small subset of these metabolites, often referred to as primary metabolites, are essential for plant life, the vast majority are specialized metabolites that are often phylogenetically restricted and have evolved as adaptations to specific abiotic and biotic environments. The bioactivities of plant specialized metabolites have led to human applications of these natural products as aromas (e.g. linalool in perfume), spices (e.g. black pepper, basil, glucosinolates), and as a source of stimulants and pharmaceuticals (e.g. caffeine, aspirin, and Taxol). Tropane alkaloids are a medicinally important class of specialized metabolite found in only a few plant families that includes cocaine in the Erythroxylaceae and scopolamine in the Solanaceae. These compounds are characterized by an eight-membered, nitrogen-containing, bicyclic ring, and their medicinal properties arise from their anticholinergic effects. The pathway leading to the biosynthesis of scopolamine in the Solanaceae is known, and elucidation of this route was facilitated by the availability of a tissue-specific transcriptome of Atropa belladonna (Deadly Nightshade) and the development of a reverse-genetics platform, using virus-induced gene silencing, for the functional screening of candidate genes. However, alkaloid chemical diversity stemming from the scopolamine pathway remains largely uncharacterized. In this dissertation, I focus on characterizing alkaloid chemical diversity generated by pathway intermediates and enzymes at three distinct points of the scopolamine pathway. First, I characterize pyrrolidine alkaloid diversity using multivariate statistics and liquid chromatography coupled to mass spectrometry (LC-MS) based metabolomics on PyKS-silenced A. belladonna roots. While the silencing of PyKS (pyrrolidine ketide synthase), a gene required for the formation of tropinone, results in an expected decrease in tropane alkaloids, it also leads to the accumulation of the N-methyl-Δ1-pyrrolinium cation. Annotation of metabolites increased in PyKS-silenced plants revealed previously unknown pyrrolidine alkaloid diversity that is generated through a non-enzymatic reaction between the N-methyl-Δ1-pyrrolinium cation and 2-O-malonylphenyllactate followed by modification of this metabolite by promiscuous enzyme activities typically associated with plant specialized metabolism. Secondly, I characterize diversity in tropane alkaloids by examining the ability of AbArAT4 (aromatic amino acid transferase 4), AbPPAR (phenylpyruvic acid reductase), AbUGT1 (UDP-glycosyltransferase 1), and AbLS (littorine synthase), enzymes that utilize phenylalanine-derived metabolites in scopolamine biosynthesis, to funnel tryptophan and tyrosine into the biosynthesis of additional tropane alkaloid aromatic esters. I show that these enzymes are promiscuous and provide support for the structural annotation of these tyrosine- and tryptophan-derived tropane aromatic esters. Lastly, I utilize gene-silencing and metabolite engineering to characterize the role of promiscuity in enzymes in the later steps of the pathway, LM (littorine mutase) and H6H (hyoscyamine-6β-hydroxylase), in generating a wealth of tropane alkaloid isomers. The use of synthetic and molecular biology tools facilitated the annotation of isomers that were difficult to resolve with traditional analytical techniques. Further elucidation of chemical diversity in this pathway has highlighted the role of nonenzymatic mechanisms and enzymatic promiscuity in shaping the metabolome and demonstrated the power of coupling traditional metabolomic tools to reverse-genetics and synthetic biology for the annotation of metabolites.
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