The Evolutionary Pattern of Cocaine and Hyoscyamine Biosynthesis Provides Strategies To Produce Tropane Alkaloids.

Cocaine and hyoscyamine are two tropane alkaloids (TA) from Erythroxylaceae and Solanaceae, respectively. These famous compounds possess anticholinergic properties that can be used to treat neuromuscular disorders. While the hyoscyamine biosynthetic pathway has been fully elucidated allowing its de novo synthesis in yeast, the cocaine pathway remained only partially elucidated. Recently, the Huang research group has completed the cocaine biosynthetic route by characterizing its two missing enzymes. This allowed the whole pathway to be transferring into Nicotiana benthamiana to achieve cocaine production. Here, besides highlighting the impact of this discovery, we discuss how TA biosynthesis evolved via the recruitment of two distinct and convergent pathways in Erythroxylaceae and Solanaceae. Finally, while enriching our knowledge on TA biosynthesis, this diversification of the molecular actors involved in cocaine and hyoscyamine biosynthesis opens perspectives in metabolic engineering by exploring enzyme biochemical plasticity that can ease and shorten TA pathway reconstitution in heterologous organisms.

Elucidation of tropane alkaloid biosynthesis in Erythroxylum coca using a microbial pathway discovery platform.

Tropane alkaloids (TAs) are heterocyclic nitrogenous metabolites found across seven orders of angiosperms, including Malpighiales (Erythroxylaceae) and Solanales (Solanaceae). Despite the well-established euphorigenic properties of Erythroxylaceae TAs like cocaine, their biosynthetic pathway remains incomplete. Using yeast as a screening platform, we identified and characterized the missing steps of TA biosynthesis in Erythroxylum coca. We first characterize putative E. coca polyamine synthase- and amine oxidase-like enzymes in vitro, in yeast, and in planta to show that the first tropane ring closure in Erythroxylaceae occurs via bifunctional spermidine synthase/N-methyltransferases and both flavin- and copper-dependent amine oxidases. We next identify a SABATH family methyltransferase responsible for the 2-carbomethoxy moiety characteristic of Erythroxylaceae TAs and demonstrate that its coexpression with methylecgonone reductase in yeast engineered to express the Solanaceae TA pathway enables the production of a hybrid TA with structural features of both lineages. Finally, we use clustering analysis of Erythroxylum transcriptome datasets to discover a cytochrome P450 of the CYP81A family responsible for the second tropane ring closure in Erythroxylaceae, and demonstrate the function of the core coca TA pathway in vivo via reconstruction and de novo biosynthesis of methylecgonine in yeast. Collectively, our results provide strong evidence that TA biosynthesis in Erythroxylaceae and Solanaceae is polyphyletic and that independent recruitment of unique biosynthetic mechanisms and enzyme classes occurred at nearly every step in the evolution of this pathway.

Tropane alkaloids from the stem bark of Erythroxylum bezerrae.

Six previously undescribed tropane alkaloids, designated as erythrobezerrines A-F, were isolated from the EtOH extract from the stem bark of Erythroxylum bezerrae Plowman. Their structures were elucidated based on the interpretation of the NMR and MS data and in some instances, confirmed by X-ray diffraction analysis. The cytotoxicity of the isolated compounds was evaluated against the cancer cell lines L929, PC-3, HCT-116, SNB-19 and NCI-H460, but only erythrobezerrine C showed moderate activity with IC50 values of 3.38 and 5.43 µM for HCT-116 and NCI-H460, respectively.

Patterns of variation in distylous traits and reproductive consequences in Erythroxylum species and populations.

PREMISE: Distylous species possess two floral morphs with reciprocal positioning of stigmas and anthers that is hypothesized to promote disassortative pollination. Theoretical models predict equal morph frequencies, but many populations depart from the expected 1:1 ratio, a pattern that often correlates with asymmetric mating between morphs and/or presence of a weak incompatibility system. Variation in reciprocity can also affect the likelihood of disassortative pollination and, hence, reproductive fitness. METHODS: We described variation in incompatibility systems and morph ratio in four Erythroxylum species to test if greater deviations from 1:1 ratios occur in populations of self-compatible species. Using adaptive inaccuracy, we described upper and lower organ reciprocity in species and populations and assessed the relationship of reciprocity to population means and coefficients of variation for fruit set to test if reciprocity could predict female reproductive success. RESULTS: Morphs occurred in 1:1 ratios in most populations of three Erythroxylum species with distylous self-incompatibility. In self-compatible E. campestre populations showed an excess of the long-styled morph, the short-styled morph, or were monomorphic for the short-styled morph. We detected deviations from reciprocity, with total inaccuracy ranging between 9.39% and 42.94%, and inaccuracy values were lowest in low organs. Across populations, we found a positive relationship between inaccuracy and the coefficient of variation of fruit set. CONCLUSIONS: Erythroxylum species showed variation in the distylous syndrome, with changes in the incompatibility system that corresponded with deviations from 1:1 morph ratio, and variation in reciprocity that correlated with variation in female reproductive fitness.

Auto-oxidation of Ent-beyer-15-en-19-al isolated from the essential oil of the heartwood of Erythroxylum monogynum Roxb.: formation of 15,16-epoxy-ent-beyeran-19-oic acid and other products.

Chemical investigation of the essential oil obtained from the heartwood of Erythroxylum monogynum Roxb. yielded three beyerene type diterpenoids ent-beyer-15-ene (1), ent-beyer-15-en-19-ol (erythroxylol A) (2) and ent-beyer-15-en-19-al (3). Ent-beyer-15-en-19-al (3) was found to be unstable at room temperature, giving rise to hitherto unknown 15,16-epoxy-ent-beyeran-19-oic acid (4). This conversion involves the auto-oxidation of a C-4 axial aldehyde group of an ent-beyer-15-ene diterpenoid with the concurrent epoxidation of the C-15 double bond. This is the first report of the auto-oxidation of an aldehyde group to a carboxylic acid group with the concurrent epoxidation of a double bond in the same compound. Further investigation of this observation under controlled conditions resulted in the isolation and identification of ent-beyer-15-en-19-oic acid (5), two new epoxy hydroperoxides, 15,16-epoxy-19-nor-ent-beyeran-4α-hydroperoxide (6a), 15,16-epoxy-18-nor-ent-beyeran-4ß-hydroperoxide (6b), and two new hydroperoxides, ent-beyer-19-nor-15-en-4α-hydroperoxide (7), ent-beyer-18-nor-15-en-4ß-hydroperoxide (8) and ent-beyer-18-nor-15-en-4ß-ol (9). Identification of these compounds was carried out by the extensive usage of spectroscopic data including 1D and 2D NMR. The acid 5 and the alcohol 9 have been reported previously as natural products from Elaeoselinum asclepium and Erythroxylum monogynum. The mechanistic basis of this auto-oxidation reaction is discussed.

Estimation of leaf area of Erythroxylum citrifolium from linear leaf dimensions / Estimativa da área foliar de Erythroxylum citrifolium a partir de dimensões lineares da folha

Erythroxylum citrifolium is a neotropical plant species recorded in all regions of Brazil. Determining leaf area is of fundamental importance to studies related to plant propagation and growth. The objective was to obtain an equation to estimate the leaf area of E. citrifolium from linear dimensions of the leaf blade (length and width). A total of 200 leaf blades were collected in Parque Estadual Mata do Pau-Ferro in the municipality of Areia, state of Paraíba, Northeast Brazil. The models evaluated were: linear, linear without intercept, quadratic, cubic, power and exponential. The best model was determined by the criteria of: high coefficient of determination (R²), low root mean square error (RMSE), low Akaike information criterion (AIC), high Willmott concordance index (d) and a BIAS index close to zero. All of the models constructed satisfactorily estimated the leaf area of E. citrifolium, with coefficients of determination above 0.9050, but the power model using the product between length and width (L*W) y = 0.5966 * LW1.0181 was the best, with the highest values of R² and d, low values of RMSE and AIC, and a BIAS index closest to zero.

Erythroxylum citrifolium é uma espécie de planta neotropical com registros em todas as regiões do Brasil. A determinação da área foliar é de fundamental importância em estudos relacionados a propagação e crescimento vegetal. O objetivo foi obter uma equação que permita estimar a área foliar de E. citrifolium a partir de dimensões lineares do limbo foliar (comprimento e largura). Foram coletados 200 limbos foliares no Parque Estadual Mata do Pau-Ferro, Areia, Paraíba, Nordeste do Brasil. Os modelos empregados foram: linear, linear sem intercepto, quadrático, cúbico, potencial e exponencial. Os critérios utilizados para escolher o melhor modelo, teve como base o maior coeficiente de determinação (R²), menor raiz do quadrado médio do erro (RMSE), menor critério de informação de Akaike (AIC), maior índice de concordância de Willmott (d) e índice BIAS mais próximo de zero. Todos os modelos construídos podem estimar satisfatoriamente a área foliar de E. citrifolium, com coeficientes determinação acima de 0,9050, porém o modelo potencial utilizando o produto entre comprimento e largura (L*W) y = 0,5966 * LW1,0181 é o mais indicado, com os maiores valores de R² e d, menores valores de RMSE e AIC, e índice BIAS mais próximo de zero.

Erythroxylum in Focus: An Interdisciplinary Review of an Overlooked Genus.

The genus Erythroxylum contains species used by indigenous people of South America long before the domestication of plants. Two species, E. coca and E. novogranatense, have been utilized for thousands of years specifically for their tropane alkaloid content. While abuse of the narcotic cocaine has impacted society on many levels, these species and their wild relatives contain untapped resources for the benefit of mankind in the form of foods, pharmaceuticals, phytotherapeutic products, and other high-value plant-derived metabolites. In this review, we describe the current state of knowledge of members within the genus and the recent advances in the realm of molecular biology and biochemistry.

Phylogenetic inference in section Archerythroxylum informs taxonomy, biogeography, and the domestication of coca (Erythroxylum species).

PREMISE OF THE STUDY: This investigation establishes the first DNA-sequence-based phylogenetic hypothesis of species relationships in the coca family (Erythroxylaceae) and presents its implications for the intrageneric taxonomy and neotropical biogeography of Erythroxylum. We also identify the closest wild relatives and evolutionary relationships of the cultivated coca taxa. METHODS: We focused our phylogenomic inference on the largest taxonomic section in the genus Erythroxylum (Archerythroxylum O.E.Schulz) using concatenation and gene tree reconciliation methods from hybridization-based target capture of 427 genes. KEY RESULTS: We show that neotropical Erythroxylum are monophyletic within the paleotropical lineages, yet Archerythroxylum and all of the other taxonomic sections from which we sampled multiple species lack monophyly. We mapped phytogeographic states onto the tree and found some concordance between these regions and clades. The wild species E. gracilipes and E. cataractarum are most closely related to the cultivated E. coca and E. novogranatense, but relationships within this "coca" clade remain equivocal. CONCLUSIONS: Our results point to the difficulty of morphology-based intrageneric classification in this clade and highlight the importance of integrative taxonomy in future systematic revisions. We can confidently identify E. gracilipes and E. cataractarum as the closest wild relatives of the coca taxa, but understanding the domestication history of this crop will require more thorough phylogeographic analysis.

Temperatures and substrates for germination and vigor ofErythroxylum pauferrense Plowman seeds

Erythroxylum pauferrense is an endemic understory plant species of the Northeast Region of Brazil. The species is of great importance to the region and so ecophysiological studies are needed for its preservation. The objective of the present study was to determine the best substrates and temperatures for testing germination and seed vigor of E. pauferrense. An experiment was performed comprising a completely randomized design in a 5 x 4 factorial scheme, with five temperature regimes (20, 25, 30, 35°C constant and 20-30°C alternating) and four types of substrates (paper, vermiculite, sand and commercial substrate). The following characteristics were evaluated: germination percentage, first germination count, germination speed index, mean germination time, seedling length and dry mass (root and shoot). Paper and vermiculite substrates combined with constant temperatures of 20, 25, 30 and alternated between 20-30°C, provide greater seed germination and vigor while 35°C reduces seed physiological quality.

Isolation, spectral characterization, molecular docking, and cytotoxic activity of alkaloids from Erythroxylum pungens O. E. Shulz.

Stem bark, root bark, and leaf extracts of Erythroxylum pungens were subjected to phytochemical analysis. N,N-dimethyltryptamine (DMT) was isolated and characterized from E. pungens roots. This unprecedented result is remarkable since no indole alkaloid has been previously reported from Erythroxylaceae so far. Eleven known tropane alkaloids were identified by their mass spectra and 3-(2-methylbutyryloxy)tropan-6,7-diol as well as 3-(2-methylbutyryloxy)nortropan-6,7-diol were isolated and characterized based on mass spectrometry, 1H, 13C, COSY, and NOESY NMR analysis. The complete NMR data are reported for the first time. Inverse Structure-based and Ligand-Based virtual screening were carried out to identify possible targets for 3-(2-methylbutyryloxy)tropan-6,7-diol. The level of cytotoxicity of this tropane alkaloid aliphatic ester was discrete with potencies on the order of 0.3-1.0 mg/mL and better results against HeLa (50% cell viability reduction). Otherwise, atropine (0.3 mg/mL), a Solanaceae tropane alkaloid, and DMT (0.5 mg/mL) from E. pungens roots impaired at 50% the cell viability against HeLa, SiHa, PC3, and 786-0. This study stimulates scientific investigation of the impact of edaphoclimatic features in a semi-arid environment on tropane alkaloid biosynthesis.

Revisión sobre el uso del mate de hoja de coca en la prevención del mal agudo de montaña / Review on the use of coca tea in prevention of acute mountain sickness

El número de turistas que viajan a zonas de gran altitud en países andinos se está  incrementando en los últimos años, con aumento concomitante del riesgo de desarrollar el mal agudo de montaña. En las zonas alto andinas el mate de hoja de coca es usado frecuentemente para la prevención de este problema, pero las evidencias no soportan esta medida; se estima que, por el contrario, podría incrementar los síntomas del cuadro clínico que se intenta controlar por lo que se sugiere la conveniencia de no incentivar su uso.

In recent years, an increasing number of tourists visiting high altitude zones and places in Andean countries are, in turn, making a concomitantgreater risk of developing acute mountain sickness. In the Andean region, coca tea is frequently used for the prevention of this condition. However, existing evidence does not seem to support such use and, on the contrary, it is estimated that it may increase the symptoms. The convenience of not to encourage the use of coca tea forthe prevention of acute mountain sickness, is suggested.

Intoxicação espontânea e experimental por folhas e frutos de Erythroxylum deciduum (cocão) em ovinos no Estado de Santa Catarina / Spontaneous and experimental poisoning by the leaves and fruits of Erythroxylum deciduum (Cocão) in sheep in the State of Santa Catarina, Brazil

Descreve-se uma enfermidade em ovinos de ocorrência sazonal, caracterizada por manifestações nervosas e morte rápida, nos municípios de Rancho Queimado e Ponte Alta do Sul, no estado de Santa Catarina. A doença foi reproduzida experimentalmente em ovinos com a administração de folhas de Erythroxylum deciduum coletados durante o verão, com doses letais a partir de 10g/kg de peso vivo. Em um único experimento com os frutos a dose de 15g/kg foi letal. O início da manifestação dos sinais clínicos, ocorreu entre 0 e 2 horas após a administração das folhas ou dos frutos e a evolução da doença foi de 10 minutos a 18 horas. Doses de até 20g/kg de folhas coletadas no período de inverno, produziram alterações clínicas discretas em dois ovinos. Os sinais clínicos foram depressão, sonolência e, quando os animais eram movimentados, incoordenação motora, desequilíbrio, quedas, dificuldade para se levantar e estação com os membros abertos. Também foi observado: salivação intensa, regurgitação, bruxismo, aumento das frequências cardíaca e respiratória. Os sinais clínicos se agravavam quando os animais eram movimentados e geralmente resultavam em morte. Os animais que morreram pelas intoxicações espontânea e experimental, não tinham alterações macroscópicas e microscópicas significativas. O corte das árvores de E. deciduum faz com que o tronco rebrote, favorecendo a ingestão da planta pelos ovinos. Este fato e a disponibilidade de frutos maduros que caem ao chão podem ser responsabilizados pela doença de ovinos com sinais nervosos e ausência de lesões macroscópicas e microscópicas, que ocorrem nos meses de verão, no Estado de Santa Catarina.

A disease in sheep with seasonal occurrence in the municipalities of Rancho Queimado and Ponte Alta do Sul, State of Santa Catarina, characterized by nervous manifestations and rapid death is described. The disease was reproduced experimentally in sheep giving the leaves of Erythroxylum deciduum collected in summer, at lethal doses from 10g/kg on. In one experiment in sheep with the fruits, the dose of 15g/kg was lethal. The onset of symptoms occurred within 2 hours after administration of the leaves, and the poisoning had a course from 10 minutes to 18 hours. Doses up to 20g/kg of leaves collected in winter caused slight signs of poisoning in two sheep. In the experiments, the clinical signs were depression and somnolence, and when the animals were moved, motor incoordination, imbalance, falls, difficulty to get up, and station with open legs; intense salivation, regurgitation, bruxism, increased heart rate and breathing was also observed. The clinical signs and death were exacerbated when the sheep were moved. The animals that died by the spontaneous and experimental poisoning did not show important gross lesions or microscopic alterations. It was concluded that the cut of the tree, allows growth of sprouts from the stems, encouraging the sheep to eat the plant. This fact and the availability of ripe fruits that fall off the trees can be made responsible for the poisoning with nervous signs of sheep, which occurs during the summer months in the state of Santa Catarina.

Duas novas espécies de Lopesia Rübsaamen (Diptera, Cecidomyiidae) do Brasil, com chave para as espécies / Two new species of Lopesia Rübsaamen (Diptera, Cecidomyiidae) from Brazil, with an identification key of species

Lopesia erythroxyli Rodrigues & Maia e Lopesia maricaensis Rodrigues & Maia, duas novas espécies de Cecidomyiidae que induzem galhas cônicas em gemas de Erythroxylum ovalifolium Peyr (Erythroxylaceae) e enrolamentos da borda da folha de Protium brasiliense (Spr.) Engl. (Burseraceae), são descritas e ilustradas (larva, pupa, macho, e fêmea) baseado em material do Rio de Janeiro, Brasil. Uma chave de segregação para as espécies conhecidas de Lopesia é dada.

Lopesia erythroxyli Rodrigues & Maia and Lopesia maricaensis Rodrigues & Maia, two new species of Cecidomyiidae that induce conical galls on bud of Erythroxylum ovalifolium Peyr (Erythroxylaceae) and on marginal leaf roll of Protium brasiliense (Spr.) Engl. (Burseraceae), are described and ilustrated (larva, pupa, male, and female) based on material from Rio de Janeiro, Brazil. A key to the known species of Lopesia is given.

Erythroxylaceae endémicas del Perú

La familia Erythroxylaceae es reconocida en el Perú por presentar un género, Erythroxylum, y 27 especies (Brako & Zarucchi, 1993; Ulloa Ulloa et al., 2004), todas arbustos y árboles. En este trabajo reconocemos cuatro especies endémicas, las cuales ocupan las regiones Desierto Cálido Tropical y Bosques Húmedos Amazónicos, entre los 150 y 720 m de altitud. Las Erythroxylaceae endémicas no se encuentran representadas en el Sistema Nacional de Áreas Naturales Protegidas por el Estado.

The Erythroxylaceae are represented in Peru by 27 species in the genus Erythroxylum (Brako & Zarucchi, 1993; Ulloa Ulloa et al., 2004), all shrubs and trees. Here we recognize four endemic species, which are found in the Tropical Costal Desert and Humid Lowland Amazonian Forests regions, between 150 and 720 m elevation. No endemic Erythroxylaceae have been recorded within Peru's protected areas system.

O gênero Erythroxylum P. Browne (Erythroxylaceae) do estado do Paraná, Brasil

An inventory of the Erythroxylum's species from the State of Paraná, Brazil, was done and the following species were found: E. ambiguum Peyr., E. amplifolium (Mart.) O.E. Schulz, E. anguifugum Mart., E. argentinum O.E. Schulz, E. buxus Peyr., E. campestre A. St.-Hil., E. cuneifolium (Mart.) O.E. Schulz, E. cuspidifolium Mart., E. deciduum A. St.-Hil., E. gonocladum (Mart.) O.E. Schulz, E. nanum A. St.-Hil., E. pelleterianum A. St.-Hil., E. microphyllum A. St.-Hil., E. myrsinites Mart. and E. suberosum A. St.-Hil.. All of then are taxonomically revised and a new synonym is presented: E. cuneifolium (Mart.) O.E. Schulz var. squarrosum O.E. Schulz as synonym of E. cuneifolium (Mart.) O.E. Schulz. Is also discussed the validity of the genus name Erythroxylon Linnaeus against Erythroxylum P. Browne.