Homolog of tocopherol C methyltransferases catalyzes N methylation in anticancer alkaloid biosynthesis

Madagascar periwinkle (Catharanthus roseus) is the sole source of the anticancer drugs vinblastine and vincristine, bisindole alkaloids derived from the dimerization of the terpenoid indole alkaloids vindoline and catharanthine. Full elucidation of the biosynthetic pathways of these compounds is a prerequisite for metabolic engineering efforts that will improve production of these costly molecules. However, despite the medical and commercial importance of these natural products, the biosynthetic pathways remain poorly understood. Here we report the identification and characterization of a C. roseus cDNA encoding an S-adenosyl-L-methionine-dependent N methyltransferase that catalyzes a nitrogen methylation involved in vindoline biosynthesis. Recombinant enzyme produced in Escherichia coli is highly substrate specific, displaying a strict requirement for a 2,3-dihydro bond in the aspidosperma skeleton. The corresponding gene transcript is induced in methyl jasmonate-elicited seedlings, along with the other known vindoline biosynthetic transcripts. Intriguingly, this unique N methyltransferase is most similar at the amino acid level to the plastidic ?-tocopherol C methyltransferases of vitamin E biosynthesis, suggesting an evolutionary link between these two functionally disparate methyltransferases

Three new polyketides from fruiting bodies of the endophytic ascomycete <i>Xylaria polymorpha</i>

<p>The isolation of three new secondary metabolites from the fruiting body of <i>Xylaria polymorpha</i> is described. The new compounds are of mixed biosynthetic origin consisting of a polyketide starter, extended with a methyl orsellinate unit and terminated hydrolytically or with an amine-containing terminal unit.</p

Mechanistic studies on norcoclaurine synthase of benzylisoquinoline alkaloid biosynthesis: An enzymatic Pictet-Spengler reaction

Norcoclaurine synthase catalyzes an asymmetric Pictet−Spengler condensation of dopamine and 4-hydroxyphenylacetaldehyde to give (S)-norcoclaurine. This is the first committed step in the biosynthesis of the benzylisoquinoline alkaloids that include morphine and codeine. In this work, the gene encoding for the Thalictrum flavum norcoclaurine synthase is highly overexpressed in Escherichia coli and the resulting His-tagged recombinant enzyme is purified for the first time. A continuous assay based on circular dichroism spectroscopy is developed and used to monitor the kinetics of the enzymatic reaction. Dopamine analogues bearing a methoxy or hydrogen substituent in place of the C-1 phenolic group were readily accepted by the enzyme whereas those bearing the same substituents at C-2 were not. This supports a mechanism involving a two-step cyclization of the putative iminium ion intermediate that does not proceed via a spirocyclic intermediate. The reaction of [3,5,6-2H]dopamine was found to be slowed by a kinetic isotope effect of 1.7 ± 0.1 on the value of kcat/KM. This is interpreted as showing that the deprotonation step causing rearomatization is partially rate determining in the overall reaction

A Stereoselective Hydroxylation Step of Alkaloid Biosynthesis by a Unique Cytochrome P450 in \u3ci\u3eCatharanthus roseus\u3c/i\u3e

Plant cytochrome P450s are involved in the production of over a hundred thousand metabolites such as alkaloids, terpenoids, and phenylpropanoids. Although cytochrome P450 genes constitute one of the largest superfamilies in plants, many of the catalytic functions of the enzymes they encode remain unknown. Here, we report the identification and functional characterization of a cytochrome P450 gene in a new subfamily of CYP71, CYP71BJ1, involved in alkaloid biosynthesis. Co-expression analysis of putative cytochrome P450 genes in the Catharanthus roseus transcriptome identified candidate genes with expression profiles similar to known terpene indole alkaloid biosynthetic genes. Screening of these candidate genes by functional expression in Saccharomyces cerevisiae yielded a unique P450-dependent enzyme that stereoselectively hydroxylates the alkaloids tabersonine and lochnericine at the 19-position of the aspidosperma-type alkaloid scaffold. Tabersonine, which can be converted to either vindoline or 19-O-acetylhörhammericine, represents a branch point in alkaloid biosynthesis. The discovery of CYP71BJ1, which forms part of the pathway leading to 19-O-acetylhörhammericine, will help illuminate how this branch point is controlled in C. roseus

A Stereoselective Hydroxylation Step of Alkaloid Biosynthesis by a Unique Cytochrome P450 in Catharanthus roseus

Plant cytochrome P450s are involved in the production of over a hundred thousand metabolites such as alkaloids, terpenoids, and phenylpropanoids. Although cytochrome P450 genes constitute one of the largest superfamilies in plants, many of the catalytic functions of the enzymes they encode remain unknown. Here, we report the identification and functional characterization of a cytochrome P450 gene in a new subfamily of CYP71, CYP71BJ1, involved in alkaloid biosynthesis. Co-expression analysis of putative cytochrome P450 genes in the Catharanthus roseus transcriptome identified candidate genes with expression profiles similar to known terpene indole alkaloid biosynthetic genes. Screening of these candidate genes by functional expression in Saccharomyces cerevisiae yielded a unique P450-dependent enzyme that stereoselectively hydroxylates the alkaloids tabersonine and lochnericine at the 19-position of the aspidosperma-type alkaloid scaffold. Tabersonine, which can be converted to either vindoline or 19-O-acetylhörhammericine, represents a branch point in alkaloid biosynthesis. The discovery of CYP71BJ1, which forms part of the pathway leading to 19-O-acetylhörhammericine, will help illuminate how this branch point is controlled in C. roseus

Functional annotation results.

<p>Proportion of <i>Camptotheca acuminata</i>, <i>Catharanthus roseus</i> and <i>Rauvolfia serpentina</i> transcripts with sequence similarity to the UniRef100 database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052506#pone.0052506-Suzek1" target="_blank">[17]</a>, <i>Arabidopsis thaliana</i> proteome (<a href="http://arabidopsis.org" target="_blank">http://arabidopsis.org</a>), and Pfam domain database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052506#pone.0052506-Punta1" target="_blank">[18]</a>.</p