The final steps of cocaine biosynthesis in the Erythroxylaceae provide insight into the biochemistry, physiology and evolution of tropane alkaloid biosynthesis

Tropane alkaloids are medicinally-valued plant secondary metabolites found in ten angiosperm plant families. Despite their medicinal value and socioeconomic impact on mankind, their biosynthesis and ecological function remains to be understood. The most biochemically investigated angiosperm family producing tropane alkaloids is the Solanaceae, predominantly known for atropine and scopolamine from belladonna (Atropa belladonna), datura (Datura stramonium), henbane (Hyoscyamus niger) and mandrake (Mandragora officinalis). The Erythroxylaceae, another important tropane alkaloid producing angiosperm family, is predominantly known for cocaine from the coca plant (Erythroxylum coca). In this thesis, I review the current state of tropane alkaloid biosynthesis in plants, their occurrence in the angiosperms and their ecological functions. Besides the Solanaceae, biochemical investigations on tropane alkaloid production in other plant families have been neglected. Therefore the last two steps of tropane alkaloid biosynthesis in E. coca were investigated. Interestingly, the penultimate step in cocaine biosynthesis in Erythroxylaceae is performed by a different family of oxidoreductase enzymes, than reported from the Solanaceae. Short chain reductases / dehydrogenases (SDR) reduce tropinone in Solanaceae and an aldo-keto reductase (AKR) reduces 2-carbomethoxy-3-tropinone in the Erythroxylaceae. The utilization of both SDR and AKR enzymes in tropane alkaloid biosynthesis in angiosperms is an example of convergent evolution. In addition, the enzyme responsible for the last step of cocaine biosynthesis, the esterification of 2-carbomethoxy-3β-tropine and benzoyl-CoA, in E. coca was characterized and belongs to the BAHD acyltransferase enzyme superfamily. The importance of biochemical investigations of plant secondary metabolite pathways is further reviewed in the context of metabolic engineering

Plant tropane alkaloid biosynthesis evolved independently in the Solanaceae and Erythroxylaceae

The pharmacologically important tropane alkaloids have a scattered distribution among angiosperm families, like many other groups of secondary metabolites. To determine whether tropane alkaloids have evolved repeatedly in different lineages or arise from an ancestral pathway that has been lost in most lines, we investigated the tropinone-reduction step of their biosynthesis. In species of the Solanaceae, which produce compounds such as atropine and scopolamine, this reaction is known to be catalyzed by enzymes of the short-chain dehydrogenase/reductase family. However, in Erythroxylum coca (Erythroxylaceae), which accumulates cocaine and other tropane alkaloids, no proteins of the short-chain dehydrogenase/reductase family were found that could catalyze this reaction. Instead, purification of E. coca tropinone-reduction activity and cloning of the corresponding gene revealed that a protein of the aldo-keto reductase family carries out this reaction in E. coca. This protein, designated methylecgonone reductase, converts methylecgonone to methylecgonine, the penultimate step in cocaine biosynthesis. The protein has highest sequence similarity to other aldo-keto reductases, such as chalcone reductase, an enzyme of flavonoid biosynthesis, and codeinone reductase, an enzyme of morphine alkaloid biosynthesis. Methylecgonone reductase reduces methylecgonone (2-carbomethoxy-3-tropinone) stereospecifically to 2-carbomethoxy-3β-tropine (methylecgonine), and has its highest activity, protein level, and gene transcript level in young, expanding leaves of E. coca. This enzyme is not found at all in root tissues, which are the site of tropane alkaloid biosynthesis in the Solanaceae. This evidence supports the theory that the ability to produce tropane alkaloids has arisen more than once during the evolution of the angiosperms

TIR domains of plant immune receptors are 2 ',3 '-cAMP/cGMP synthetases mediating cell death

2',3'-cAMP is a positional isomer of the well-established second messenger 3',5'-cAMP, but little is known about the biology of this noncanonical cyclic nucleotide monophosphate (cNMP). Toll/interleukin-1 receptor (TIR) domains of nucleotide-binding leucine-rich repeat (NLR) immune receptors have the NADase function necessary but insufficient to activate plant immune responses. Here, we show that plant TIR proteins, besides being NADases, act as 2',3'-cAMP/cGMP synthetases by hydrolyzing RNA/DNA. Structural data show that a TIR domain adopts distinct oligomers with mutually exclusive NADase and synthetase activity. Mutations specifically disrupting the synthetase activity abrogate TIR-mediated cell death in Nicotiana benthamiana (Nb), supporting an important role for these cNMPs in TIR signaling. Furthermore, the Arabidopsis negative regulator of TIR-NLR signaling, NUDT7, displays 2',3'-cAMP/cGMP but not 3',5'-cAMP/cGMP phosphodiesterase activity and suppresses cell death activity of TIRs in Nb. Our study identifies a family of 2',3'-cAMP/cGMP synthetases and establishes a critical role for them in plant immune responses