A chemically diverse set of specialized flavorings, fragrances, and medicinal metabolites are produced by plants to modulate their interactions with pollinators, herbivores, and other biotic and abiotic stresses. Tropane alkaloids are one group of these specialized metabolites which are produced in phylogenetically distinct plant families and include the narcotic cocaine and the pharmaceutical compounds hyoscyamine and scopolamine. Scopolamine is produced in the roots of plants in the Solanaceae family and serves as the precursor to semi-synthetic tropane pharmaceuticals. A set of eleven tissue-specific transcriptomes was generated from the tender perennial Solanaceous plant Atropa belladonna, deadly nightshade, to fill gaps in the biosynthetic pathway from putrescine to scopolamine. This dissertation describes the identification of enzymes which complete three missing portions of the scopolamine biosynthetic pathway. These are the biosynthesis of tropinone, the diversion of phenylalanine into production of phenyllactic acid, and the activation and conjugation of phenyllactic acid with tropine to form littorine, a late pathway precursor to scopolamine. Tropinone is the first metabolite in this pathway with the characteristic 8-azabicyclo[3.2.1]octane tropane core. This pharmacore was synthesized in a classic biomimetic chemical synthesis, but the mechanism for tropinone biosynthesis has remained an open question in all species which produce these compounds. The A. belladonna lateral root transcriptome revealed that tropinone biosynthesis proceeds through an atypical polyketide synthase which uses an imine as its starter, and that the polyketide is cyclized to tropinone by a cytochrome P450. Tropic acid, the specialized acyl group for both hyoscyamine and scopolamine, is produced from the primary metabolite phenylalanine through the intermediate phenyllactic acid. Aromatic aminotransferases are one route by which amino acids can be dedicated to specialized metabolism. Six aromatic aminotransferases are present in the A. belladonna transcriptome, and one of these, ArAT4, is a root-specific phenylalanine aminotransferase required for biosynthesis of phenyllactic acid and ultimately, scopolamine. In contrast to other aminotransferases which equilibrate multiple amino acids, this enzyme is highly directional for the consumption of phenylalanine and production of tyrosine. Littorine, a precursor of hyoscyamine and scopolamine, is the ester of tropine and phenyllactic acid. The mechanism for phenyllactic acid activation and esterification have remained as an open question of tropane alkaloid biosynthesis since the discovery of littorine. Two routes exist for activation and conjugation in plants, through either coenzyme A thioesters or glucose esters. In A. belladonna, littorine biosynthesis proceeds through a glucose ester of phenyllactic acid produced by a glucosyltransferase, UGT84A27, and an acyltransferase, LITTORINE SYNTHASE. This contrasts with cocaine acylation in the Erythroxylaceae, which uses a different route highlighting the repeated, independent origin of tropane alkaloid biosynthesis in plants. The enzymes identified in this dissertation have completed three missing sections of the tropane alkaloid biosynthetic pathway in A. belladonna, resulting in a nearly complete pathway, suitable for engineering.
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