ORCID IDs: 0000-0003-1007-4700 (J.J.); 0000-0002-4865-3938 (J.C.D.).The esterification of methylecgonine (2-carbomethoxy-3b-tropine) with benzoic acid is the final step in the biosynthetic pathway leading to the production of cocaine in Erythoxylum coca. Here we report the identification of a member of the BAHD family of plant acyltransferases as cocaine synthase. The enzyme is capable of producing both cocaine and cinnamoylcocaine via the activated benzoyl-or cinnamoyl-Coenzyme A thioesters, respectively. Cocaine synthase activity is highest in young developing leaves, especially in the palisade parenchyma and spongy mesophyll. These data correlate well with the tissue distribution pattern of cocaine as visualized with antibodies. Matrix-assisted laser-desorption ionization mass spectral imaging revealed that cocaine and cinnamoylcocaine are differently distributed on the upper versus lower leaf surfaces. Our findings provide further evidence that tropane alkaloid biosynthesis in the Erythroxylaceae occurs in the above-ground portions of the plant in contrast with the Solanaceae, in which tropane alkaloid biosynthesis occurs in the roots.

The indole ascarosides (icas) represent a highly potent class of nematode-derived modular signalling components that integrate structural inputs from amino acid, carbohydrate, and fatty acid metabolism. Comparative analysis of the crude exo-metabolome of hermaphroditic Caenorhabditis briggsae using a highly sensitive mass spectrometric screen reveals an indole ascaroside blend dominated by two new components. The structures of isolated icas#2 and icas#6.2 were determined by NMR spectroscopy and confirmed by total synthesis and chemical correlation. Low atto- to femtomolar amounts of icas#2 and icas#6.2 act in synergism to attract males indicating a function as sex pheromone. Comparative analysis of 14 Caenorhabditis species further demonstrates that species-specific indole ascaroside biosynthesis is highly conserved in the Elegans group. Functional characterization of the dominating indole ascarosides icas#2, icas#3, and icas#9 reveals a high degree of species-specificity and considerable variability with respect to gender-specificity, thus, confirming that indole ascarosides modulate different biological functions within the Elegans group. Although the nematode response was usually most pronounced towards conspecific signals, Caenorhabditis brenneri, the only species of the Elegans group that does not produce any indole ascarosides, exhibits a robust response to icas#2 suggesting the potential for interspecies interactions.

Nematodes such as the model organism Caenorhabditis elegans produce various homologous series of l-ascarylose-derived glycolipids called ascarosides, which include several highly potent signals in intra and interspecies communication as well as cross-kingdom interactions. Given their low concentrations and large number of structurally similar components, mass spectrometric screens based on high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) are commonly employed for ascaroside detection and quantification. Here, we describe a complementary gas chromatography-electron ionization mass spectrometry (GC-EIMS) screen that utilizes an ascarylose-derived K1-fragment ion signal at m/z 130.1 [CHOSi] to highlight known as well as yet unidentified ascaroside components in TMS-derivatized crude nematode exometabolome extracts. GC-EIMS-based ascaroside profiling of wild-type and mutant C. elegans facilitates the analysis of all basic ascarosides using the same ionization technique while providing excellent resolution for the complete homologous series with side chains ranging from 3 to 33 carbons. Combined screening for m/z 130.1 along with side chain-specific J1 [M - 173] and J2 [M - 291] fragment ions, as well as additional characteristic marker ions from α-cleavage, enables convenient structure assignment of ca. 200 components from wild-type and peroxisomal β-oxidation mutants including (ω - 1)-linked acyl, enoyl, β-hydroxyacyl, and 2-ketoalkyl ascarosides along with their (ω)-linked or α-methyl isomers and ethanolamide derivatives, as well as 2-hydroxyalkyl ascarosides. Given the widespread availability of GC-MS and its increasing popularity in metabolomics, this method will promote the identification of ascarosides in C. elegans and other nematodes.

Chemical communication in nematodes such as the model organism Caenorhabditis elegans is modulated by a variety of glycosides based on the dideoxysugar l-ascarylose. Comparative ascaroside profiling of nematode exometabolome extracts using a GC-EIMS screen reveals that several basic components including ascr#1 (asc-C7), ascr#2 (asc-C6-MK), ascr#3 (asc-ΔC9), ascr#5 (asc-ωC3), and ascr#10 (asc-C9) are highly conserved among the Caenorhabditis. Three novel side chain hydroxylated ascaroside derivatives were exclusively detected in the distantly related C. nigoni and C. afra. Molecular structures of these species-specific putative signaling molecules were elucidated by NMR spectroscopy and confirmed by total synthesis and chemical correlations. Biological activities were evaluated using attraction assays. The identification of (ω)- and (ω − 2)-hydroxyacyl ascarosides demonstrates how GC-EIMS-based ascaroside profiling facilitates the detection of novel ascaroside components and exemplifies how species-specific hydroxylation of ascaroside aglycones downstream of peroxisomal β-oxidation increases the structural diversity of this highly conserved class of nematode signaling molecules.

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