John D'Auria Seminar

Thursday, February 15, 2018 - 4:10pm to 5:00pm
Event Type: 

Photo courtesy of Texas Tech University  (Photo courtesy of Texas Tech University)

Department of Chemistry - Texas Tech University


Host:  Charles Stewart


"The Evolution of Tropane Alkaloid Biosynthesis in the Erythroxylaceae"

Tropane alkaloids (TAs) represent a major class of plant derived secondary metabolites known to occur commonly in the Solanaceae family but also reported from the families Convolvulaceae, Proteaceae, Rhizophoraceae and Erythroxylaceae.   These compounds are believed to serve a defensive roll for the plant and have also been exploited by humans for their pharmaceutical properties.  Since most of the information on tropane alkaloid biosynthesis comes from species of the Solanaceae, we have begun to investigate the coca plant, Erythroxylum coca (Erythroxylaceae), to determine if the pathway of biosynthesis is similar in this phylogenetically distant family. E. coca accumulates up to 1% cocaine per leaf dry weight.  Using high throughput transcriptome sequencing in addition to classical biochemical techniques, we have been able to isolate and characterize the genes and enzymes responsible for the penultimate reduction step yielding 2-carbomethoxy-3-tropinone as well as the final acylation reaction which yields cocaine and cinnamoyl cocaine in E. coca.  Both proteins are highly abundant in the spongy mesophyll and palisade parenchyma, the same tissue in which tropane alkaloids are stored.   During the course of our studies, we have been able to ascertain that the ability to produce tropane alkaloids in plants is a polyphyletic trait, having been independently evolved at least more than once throughout the course of evolutionary history.

In addition, attempts to metabolically engineer tropane alkaloids are hampered by the lack of knowledge of the required biosynthetic steps.  It is therefore essential to fully understand the underlying enzymatic mechanisms in order to achieve the goals of metabolic engineering with the ultimate goal of large-scale production of these medicinally important compounds. Current data supports the hypothesis that a Type III Polyketide synthase is responsible for the extension off the first ring and ultimate formation of the second ring in the bicyclic tropane nucleus. Several E. coca transcriptomes contain polyketide synthase-like sequences expressed in biosynthetically relevant organs. Enzyme assays performed using one candidate protein produced heterologously in the yeast Pichia pastoris yielded 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoate, the predicted non-cylclized polyketide enzyme product.  Our current proposed mechanism for this reaction predicts a novel PKS activity arising from the attack of the PKS active site cysteine on the carbon of the imine group in N-methylpyrrolinium to initiate the extension reaction. We are continuing with the molecular and biochemical characterization of these genes and their encoded enzymes.