Biochemistry and Evolution of Tropane Alkaloid Biosynthesis

Dr. John D'Auria

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. I have 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).  I am supervising the diplom thesis of my student, Gregor Schmidt regarding the characterization of one of these BAHD members that we have identified as having cocaine synthase activity.  Gregor has 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, I am also actively pursuing what enzymes 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.  With the aid of several undergraduate students, I am 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 all of our tools that we have thus far developed for E. coca to develop this system as a model for benzoic acid biosynthesis.

Biochemical and Biotechnological Approaches for Elucidating the Tropane Alkaloid Biosynthetic Pathway in E. coca

Dr. Teresa Docimo

One of the theoretical routes to the production of tropane alkaloids is through the amino acids arginine and ornithine via their respective decarboxylase enzymes.  Our research has identified that E. coca does indeed have genes which encode arginine and ornithine decarboxylase (ADC and ODC). I have successfully characterized ODC and ADC on the biochemical and molecular level.
Current research is now focused on ascertaining whether one or both of these enzymes play a critical role in tropane alkaloid biosynthesis. To do this, I am employing a biosynthetic approach with labelled glucose feeding and 13CO2 to map the patterns of incorporation of primary metabolites such as amino acids into the core tropane ring of cocaine. Furthermore, I am establishing a coca tissue culture system as well as developing a virus induced gene silencing (VIGS) protocol to provide more tools to study gene function and regulation in E. coca.

The diagram below shows the first putative biosynthetic steps in cocaine biosynthesis.

 

Characterization of Enzymes Involved in Tropane Alkaloid Biosynthesis in E. coca

The penultimate biosynthetic step in making cocaine is the reduction of 2-carbomethoxy-3-tropinone (methylecgonone) to 2-carbomethoxy-3-tropine (methylecgonine). Discovery of the enzyme performing this reaction was not possible by homology searches with tropinone reductases from the plant family Solanaceae. Instead, the amino acid sequence of the enzyme was determined by a classical biochemical approach (purifying the enzyme from a crude plant extract, 2D SDS-PAGE and protein sequencing). The isolated protein revealed that the methylecgonone reductase enzyme is not similar to previously characterized tropinone reductases found in other tropane-producing plant families. The reductase enzyme is able to reduce tropinone and methylecgonone stereospecifically by forming a 3β-OH group on the tropane ring.

Further research goals include the localization of this enzyme in plant tissue by immunohistochemistry and transcript localization via in situ hybridization. The aim of my PhD thesis is to further investigate aspects of methylecgonone reductase and to continue in the isolation of enzymes involved in cocaine biosynthesis.

 

Cocaine Storage in E. coca

 

Cocaine content in E.coca leaves can reach amounts over 1% dry weight. This fact, along with its toxic character to herbivores, led us to consider that it should be stored in a compartmentalized way. The interaction with chlorogenic acid (CGA) forms the basis of this hypothesis.

1H NMR and UV-Vis spectroscopy measurements have already proved this interaction and shown that it focuses on the aromatic part of both molecules. Ongoing immunohistochemistry experiments also appear to confirm its compartmentalization.

In addition, one of the eight BAHD acyltransferases identified in E.coca was found to be EcHQT, an enzyme responsible for the synthesis of coumaroylquinic acid, precursor of CGA. This enzyme was characterised.

Further research on this project aims to study the interaction CGA:Cocaine in vivo, for what different histolocalization as well as MALDI Imaging techniques have been proposed.

The roles of polyamines in the beginning steps of tropane alkaloid biosynthesis in Erythroxylum coca

Franziska Dolke

Formation of the pyrrolinium ring of tropane alkaloids in Erythroxylum coca is not clearly understood.  It is postulated that the five membered ring is formed from putrescine via a putrescine N-methyltransferase (PMT) to attain N-methyl putrescine.  Putrescine N-methyltransferase belongs to the spermidine synthase family because PMTs and spermidine synthases (SPDS) share similarities of the amino acid sequence.   In the subsequent step, putrescine is oxidized and then spontaneously cyclized to yield the N-methyl pyrrolinium ring. Interestingly, an intensive biochemical and molecular search has yet to reveal an active PMT from E. coca.

The aim of my diploma thesis is to synthesize labeled polyamine derivatives which will be then fed to E. coca plants. The objective of this precursor feeding is to study the incorporation pattern of different polyamine derivatives which are possible preliminary steps of the pyrrolinium ring formation. I am also interested in the enzyme which catalyzes the methylation of the polyamine.

Retrobiosynthetic study of the early intermediate steps of the cocaine biosynthetic pathway in Erythroxylum coca

Derek Mattern

My project is looking at the early steps of cocaine biosynthesis in Erythroxylum coca. In the group’s goal of elucidating the enzymatic and chemical nature of the pathway, this project is attempting to identify and verify the starting intermediates involved.

Radiolabeled experiments performed on plants found in the Solanaceae have shown that the first portion of the tropane ring is formed via N-methyl-putrescine. The enzyme responsible for N-methyl-putrescine, putrescine methyltransferase, is ubiquitously found throughout all members of the Solanaceae. Interestingly, intensive biochemical and molecular searches have yet to discover this enzyme or its activity in E. coca. This raises important evolutionary questions about the polyphyletic origins of the tropane alkaloid biosynthetic pathway.

A retrobiosynthetic approach will be used by looking at the different isotopologue patterns in proteinogenic amino acids and the tropane ring. 13CO2 will be fed to the plants for a certain period of time, metabolites and intermediates of interest (amino acid separation and tropane alkaloid extraction) will be extracted and then analyzed to determine the different isotopologue patterns by NMR.