The Pictet-Spengler reaction, which yields either a β-carboline or a tetrahydroquinoline product from an aromatic amine and an aldehyde, is widely utilized in plant alkaloid biosynthesis. Here we deconvolute the role that the biosynthetic enzyme strictosidine synthase plays in catalyzing the stereoselective synthesis of a β-carboline product. Notably, the rate-controlling step of the enzyme mechanism, as identified by the appearance of a primary kinetic isotope effect (KIE), is the rearomatization of a positively charged intermediate. The KIE of a nonenzymatic Pictet-Spengler reaction indicates that rearomatization is also rate-controlling in solution, suggesting that the enzyme does not significantly change the mechanism of the reaction. Additionally, the pH dependence of the solution and enzymatic reactions provides evidence for a sequence of acid-base catalysis steps that catalyze the Pictet-Spengler reaction. An additional acid-catalyzed step, most likely protonation of a carbinolamine intermediate, is also significantly rate controlling. We propose that this step is efficiently catalyzed by the enzyme. Structural analysis of a bisubstrate inhibitor bound to the enzyme suggests that the active site is exquisitely tuned to correctly orient the iminium intermediate for productive cyclization to form the diastereoselective product. Furthermore, ab initio calculations suggest the structures of possible productive transition states involved in the mechanism. Importantly, these calculations suggest that a spiroindolenine intermediate, often invoked in the Pictet-Spengler mechanism, does not occur. A detailed mechanism for enzymatic catalysis of the β-carboline product is proposed from these data.

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-acetylhör-hammericine, 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. Cytochrome P450 enzymes (P450s)5 play a key role in the development and survival of plants (1). P450s participate in the biosynthesis of a wide variety of compounds throughout primary and specialized metabolism, including fatty acids, terpenoids, phenylpropanoids, cyanogenic glucosides, glucosinolates, and alkaloids. Notably, plants have an unusually large number of P450 genes compared with prokaryotes and other eukaryotic organisms. Furthermore, plant P450s are typically stringently substrate-specific enzymes that catalyze highly regio-and stereoselective transformations (2, 3). The catalytic functions of most plant P450s are unknown. Identifying a P450 that catalyzes a specific biosynthetic transformation poses a challenge due to the homology shared by P450 genes and the typical lack of correlation between primary structure and catalytic function (4).The medicinal plant Catharanthus roseus is currently the sole source of two anticancer agents subject to widespread clinical use, the bisindole alkaloids vinblastine and vincristine (5). In C. roseus, the alkaloid tabersonine can be transformed into the bisindole precursor vindoline, in aerial organs, or 19-Oacetylhörhammericine in roots (see Fig. 1) (5, 6). Although hydroxylation at the 16-position of tabersonine ultimately leads to vindoline, tabersonine can be converted by a P450-dependent 6,7-epoxidase to form lochnericine (7-9) or a putative P450-dependent 19-hydroxylase to form 19-hydroxytabersonine (10). Both lochnericine and 19-hydroxytabersonine are proposed intermediates in 19-O-acetylhörhammericine biosynthesis (Fig. 1), and minovincinine 19-hydroxy-O-acetyltransferase is the only gene in this pathway for which a cognate cDNA has been isolated and characterized (6). Although alkaloid biosynthesis in C. roseus involve...

Alkaloid profiles for 81 individual mantellid frogs, Mantella baroni (Boulenger 1988) (N = 19), M. bernhardi (N = 51), and M. madagascariensis (Grandidier 1877) (N = 11), from six different populations from Madagascar were examined. Marked individual differences in alkaloid composition (number, type, and amount) were observed between different species and between populations of the same species. Disjunct populations of each of the three species differed significantly in alkaloid composition. Sympatric populations of M. baroni and M. madagascariensis also differed significantly in alkaloid composition. In M. bernhardi, differences in alkaloid composition were marginally associated with different sexes. A total of 111 alkaloids, including isomers, were detected in analysis of the individuals from the three species. The majority (47%) appear likely to be obtained from dietary mites, whereas many of the others (18%) are presumed to be from ants, and a few (4%) are from millipedes. Putative dietary sources for the remaining alkaloids are generally unknown, but beetles are probably the source of at least some of the tricyclic alkaloids (6%). In addition, alkaloid compositions from extracts of groups of individuals from five additional populations of M. baroni and from one population of M. bernhardi (Vences et al. 1994) and one population of M. cowanii (Boulenger 1882) were examined. An additional 50 alkaloids, including isomers, were detected in the combined samples, bringing the total number of alkaloids identified from these four species of mantellid frogs to 161. Alkaloid compositions in mantellid poison frogs are diverse and highly dependent on geographic location that appear to be largely determined by the nature and availability of alkaloid-containing prey items.

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