Biochemistry and Evolution of Tropane Alkaloid Biosynthesis

Tropane alkaloids represent a major class of plant-derived secondary metabolites known to occur in the Solanaceae family but are also present in the families Convolvulaceae, Proteaceae, Rhizophoraceae and Erythroxylaceae. The core defining structure of tropane alkaloids is an 8-azabicyclo[3.2.1] octane nucleus. The diversity of tropane alkaloids is achieved by elaboration of this core through different types of modifications. The genus Erythroxylum (family Erythroxylaceae) contains approximately 230 species with ranges spread throughout the tropics including South America and Madagascar.

 

Erythroxylum coca and Erythroxylum novogranatense are the most widely used species for the production of cocaine. Very little is known as to the biological and ecological roles that cocaine and other tropane alkaloids play in plants. Their anti-cholinergic properties argue strongly in favor of deterrent activity against herbivores. We have begun molecular and biochemical studies in order to elucidate the biochemical steps which lead to the production of tropane alkaloids in E. coca plants.

 

The terminal step in the production of cocaine or other tropane related esters is thought to be the formation of the acyl ester via the action of an acyltransferase enzyme. In the case of cocaine, this acyltransferase utilizes the substrates methylecgonine and benzoyl CoenzymeA to produce cocaine and free CoA. Our lab has been working for several years on a plant specific family of acyltransferases commonly referred to as the BAHD acyltransferases. Thus far, more than 8 BAHD acyltransferases have been isolated from Erythroxylum coca (E. coca). Recent results show one of these BAHD members exhibits cocaine synthase activity. Members of my group have successfully developed an LC-MS based ‘realtime' enzyme assay for cocaine synthase in order to obtain very accurate kinetic data for characterization studies. We are also using antibodies made against the whole purified protein in order to perform immunoprecipitation and immunohistochemical studies.

In addition to the study of the role of acyltransferases in E. coca, members of the D’Auria lab are also actively pursuing the enzymes that are involved in forming the first and second rings of the tropane core. Most theories to date suggest that the precursor compound is most likely the mono-methylated polyamine putrescine. Benjamin Chavez, a PhD. student in the D’Auria lab, is characterizing the properties of several polyamine synthases that are similar to putrescine methyltransferase and spermine/spermidine synthases. We are also interested in the origins of the benzoic acid portion of cocaine and are combining the tools that we have thus far developed for E. coca to develop this system as a model for benzoic acid biosynthesis.