Vinorine synthase is an acetyltransferase that occupies a central role in the biosynthesis of the antiarrhythmic monoterpenoid indole alkaloid ajmaline in the plant Rauvolfia. Vinorine synthase belongs to the benzylalcohol acetyl-, anthocyanin-O-hydroxy-cinnamoyl-, anthranilate-N-hydroxy-cinnamoyl/benzoyl-, deacetylvindoline acetyltransferase (BAHD) enzyme superfamily, members of which are involved in the biosynthesis of several important drugs, such as morphine, Taxol, or vindoline, a precursor of the anti-cancer drugs vincaleucoblastine and vincristine. The x-ray structure of vinorine synthase is described at 2.6-Å resolution. Despite low sequence identity, the two-domain structure of vinorine synthase shows surprising similarity with structures of several CoA-dependent acyltransferases such as dihydrolipoyl transacetylase, polyketide-associated protein A5, and carnitine acetyltransferase. All conserved residues typical for the BAHD family are found in domain 1. His 160 of the HXXXD motif functions as a general base during catalysis. It is located in the center of the reaction channel at the interface of both domains and is accessible from both sides. The channel runs through the entire molecule, allowing the substrate and co-substrate to bind independently. Asp 164 points away from the catalytic site and seems to be of structural rather than catalytic importance. Surprisingly, the DFGWG motif, which is indispensable for the catalyzed reaction and unique to the BAHD family, is located far away from the active site and seems to play only a structural role. Vinorine synthase represents the first solved protein structure of the BAHD superfamily.The acyl-CoA-dependent BAHD 1 superfamily is a fast growing enzyme family that has only recently been defined (1). The name BAHD is coined from the first four enzymes of the family isolated from plant species. The members of this family play an important role in the biosynthesis of a variety of secondary metabolites. The family might become significantly larger in the near future because ϳ70 BAHD-related genes have been identified recently in the Arabidopsis genome (2), and in most cases, their biochemical function still needs to be explored. Several BAHD members occurring in medicinal plants and fungi play very specific metabolic roles in biosynthetic pathways. The most prominent members are, for instance, those participating in the biosynthesis of the Catharanthus alkaloid vindoline (3), a precursor of the anti-cancer drugs vincaleucoblastine and vincristine, the Papaver alkaloid morphine (4), the diterpenoid alkaloid Taxol (5-7), anthocyanins (8 -10) as well as some phytoalexins (11), and enzymes involved in floral scent (12).A well-characterized enzyme of this family is vinorine synthase (VS; EC 2.3.1.160), which is of central importance in the endogenous formation of monoterpenoid indole alkaloids of the ajmalan type in the plant genus Rauvolfia. The synthase is located in the middle of the complex biosynthetic pathway that starts with tryptamine and the monoterpene s...

The enzyme strictosidine synthase (STR1) from the Indian medicinal plant Rauvolfia serpentina is of primary importance for the biosynthetic pathway of the indole alkaloid ajmaline. Moreover, STR1 initiates all biosynthetic pathways leading to the entire monoterpenoid indole alkaloid family representing an enormous structural variety of ;2000 compounds in higher plants. The crystal structures of STR1 in complex with its natural substrates tryptamine and secologanin provide structural understanding of the observed substrate preference and identify residues lining the active site surface that contact the substrates. STR1 catalyzes a Pictet-Spengler-type reaction and represents a novel six-bladed b-propeller fold in plant proteins. Structure-based sequence alignment revealed a common repetitive sequence motif (three hydrophobic residues are followed by a small residue and a hydrophilic residue), indicating a possible evolutionary relationship between STR1 and several sequence-unrelated six-bladed b-propeller structures. Structural analysis and site-directed mutagenesis experiments demonstrate the essential role of Glu-309 in catalysis. The data will aid in deciphering the details of the reaction mechanism of STR1 as well as other members of this enzyme family.

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Strictosidine glucosidase (SG) is an enzyme that catalyses the second step in the biosynthesis of various classes of monoterpenoid indole alkaloids. Based on the comparison of cDNA sequences of SG from Catharanthus roseus and raucaffricine glucosidase (RG) from Rauvolfia serpentina, primers for RT-PCR were designed and the cDNA encoding SG was cloned from R. serpentina cell suspension cultures. The active enzyme was expressed in Escherichia coli and purified to homogeneity. Analysis of its deduced amino-acid sequence assigned the SG from R. serpentina to family 1 of glycosyl hydrolases. In contrast to the SG from C. roseus, the enzyme from R. serpentina is predicted to lack an uncleavable N-terminal signal sequence, which is believed to direct proteins to the endoplasmic reticulum. The temperature and pH optimum, enzyme kinetic parameters and substrate specificity of the heterologously expressed SG were studied and compared to those of the C. roseus enzyme, revealing some differences between the two glucosidases. In vitro deglucosylation of strictosidine by R. serpentina SG proceeds by the same mechanism as has been shown for the C. roseus enzyme preparation. The reaction gives rise to the end product cathenamine and involves 4,21-dehydrocorynantheine aldehyde as an intermediate. The enzymatic hydrolysis of dolichantoside (Nb-methylstrictosidine) leads to several products. One of them was identified as a new compound, 3-isocorreantine A. From the data it can be concluded that the divergence of the biosynthetic pathways leading to different classes of indole alkaloids formed in R. serpentina and C. roseus cell suspension cultures occurs at a later stage than strictosidine deglucosylation.Keywords: strictosidine b-D-glucosidase; heterologous expression; Rauvolfia serpentina; ajmaline; indole alkaloid biosynthesis.Elucidation of the biosynthesis of natural plant products has been a matter of investigation for over half a century. Although there have been major efforts in the field, only very few biosynthetic pathways have been delineated in detail at the enzymatic level. Knowing the enzymes involved is, however, a prerequisite for understanding the biosynthetic mechanisms. The next steps are to search for the participating genes and to clarify the regulation of product synthesis, with the aim of influencing the biosynthesis on a rational basis. The best known pathways comprise those of the flavonoid biosynthesis [1,2], the isoquinoline alkaloid formation [3,4] and the biosynthesis of indole alkaloids [5,6].The key intermediate in the biosynthesis of all monoterpenoid indole alkaloids is the glucoalkaloid strictosidine [7][8][9][10]. It is formed by condensation of tryptamine, the decarboxylation product of tryptophan, and the monoterpene secologanin catalysed by the enzyme strictosidine synthase (SS) [11]. The biosynthetic pathways leading to different classes of indole alkaloids branch off somewhere downstream of strictosidine. The first point where this divergence may take place is the deglucosylation of strictos...

All the major reactions taking part in the biosynthesis of ajmaline in cell suspension cultures of the Indian medicinal plant Rauvolfia have now been established at the enzyme level. Of the well investigated 10 steps six of the involved enzymes have been functionally cloned. These are strictosidine synthase (STR), strictosidine glucosidase (SG), polyneuridine aldehyde esterase (PNAE), vinorine synthase (VS), cytochrome P450 reductase (CPR) and acetylajmalan acetylesterase (AAE). Because the cDNAs of these enzymes are now known their detailed molecular analysis became attainable for the first time. Some of these enzymes such as STR, SG or VS could be produced in E. coli at a preparative scale resulting in mg amounts of pure enzymes. They also have been crystallized and their preliminary X-ray analyses were published recently. It is only a matter of time that their molecular structure and the mechanisms of the catalyzed reactions will be elucidated. Of the soluble enzymes vomilenine reductase and 1.2-dihydrovomilenine reductase remain to be heterologously expressed. Appropriate cDNA clones have recently been isolated. What membrane bound enzymes of this pathway is concerned cloning could not be achieved up to now. Our future strategy is to purify these enzymes first and to use the "reverse genetic" approach as we did for the soluble enzymes. The sarpagine bridge enzyme (SBE), vinorine hydroxylase (VH) and norajmaline N-methyltransferase (NAMT) are the only enzymes which remain as the major candidates for expression studies in order to express heterologously the complete ajmaline biosynthetic pathway.