The molecular architecture of major enzymes from ajmaline biosynthetic pathway

Abstract

The biosynthetic pathway leading to the monoterpenoid indole alkaloid ajmaline in Rauvolfia serpentiin serpentina is one of the most studied in the field of natural product biosynthesis. Ajmaline has a complex structure which is based on a six-membered ring system harbouring nine chiral carbon atoms. There are about fifteen enzymes involved, including some involving the side reactions of the ajmaline biosynthetic pathway. All enzymes exhibit pronounced substrate specificity. In the recent years isolation and sequencing of their cDNAs has allowed a detailed sequence analysis and comparison with functionally related and occasionally un-related enzymes. Site-directed mutations of several of the ajmaline-synthesizing enzymes have been performed and their catalytic residues have been identified. Success with overexpression of the enzymes was an important step for their crystallization and structural analysis by X-ray crystallography. Crystals with sufficient resolution were obtained from the major enzymes of the pathway. Strictosidine synthase has a 3D-structure with a six-bladed bpropeller fold the first time such a fold found in the plant kingdom. Its ligand complexes with tryptamine and secologanin, as well as structure-based sequence alignment, indicate a possible evolutionary relationship to several primary sequence-unrelated structures with this fold. The structure of strictosidine glucosidase was determined and its structure has as a (b/a) 8 barrel fold. Vinorine synthase provides the first 3D structure of a member of BAHD enzyme super-family. Raucaffricine glucosidase involved in a side-route of ajmaline biosynthesis has been crystallized. The ajmaline biosynthetic pathway is an outstanding example where many enzymes 3D-structure have been known and where there is a real potential for protein engineering to yield new alkaloid.

Medicinally important secondary metabolites in recombinant microorganisms or plants: Progress in alkaloid biosynthesis

“…This topic will be explored in more detail for major alkaloid groups and the relevant newer literature is summarized in the following. Earlier and recent work has been summarized in [40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55]. (Table 1).…”

“…In the last 20 years, an impressive number of genes of secondary metabolism has been isolated (see next Sections). Several of the genes could be expressed in recombinant microorganisms and plants (reviewed in [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39] The search for the enzymes involved in the biosynthesis of alkaloids is an ongoing process and so far has been successful (at least partially) for a number of alkaloidal groups, such as morphinane-, protoberberine-, monoterpene indole-, Taxus-, ergot-, purine-, tropane-, Nicotiana-, pyrrolizidine and furanoquinoline alkaloids (see reviews [40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55]). However, considering the high structural diversity of alkaloids and other SM, much work still needs to be done until the picture is complete.…”

“…Monoterpene indole alkaloids Monoterpene indole alkaloids are especially abundant in Apocynaceae and contain several drugs of medicinal importance, such as the dimeric vinblastine and vincristine from Catharanthus roseus (important anticancer drugs), reserpine, ajmaline, ajmalicine, strychnine and yohimbine. Recent reviews of the biosynthesis of monoterpene indole alkaloids, which combine tryptamine and a monoterpene (secologanin) in their skeleton, have recently been published[47,52,55,87,88]. Strictosidine synthase (STR) is the key enzyme of this pathway…”

Molecular cloning and characterization of strictosidine synthase, a key gene in biosynthesis of mitragynine from Mitragyna speciosa

“…The study on StrMs1 protein structure enables us to predict its active site and substrate binding site in order to understand reaction mechanism. It is also useful in reconstruction of many other STR variants that can react with more substrates to produce different alkaloids (Ma et al, 2006) such as in the study of the crystal structure of STR of R. serpentina by Stöckigt et al (2007). As the crystal structure of STR from R. serpentina is already known, the structure of StrMs1 was predicted using STR R. serpentina as the template where the STR protein of R. serpentina has 60% identity with StrMs1.…”

Secondary Metabolites: Deterring Herbivores

“…The pathways leading to most groups of secondary metabolites that are listed in Table 1 have been elucidated and many of the enzymes involved have been purified and studied in detail (Conn, 1981;Luckner, 1990;Facchini, 2001;Marasco and Schmidt-Dannert, 2007;Memelink, 2005;Oksman-Caldentey et al, 2007;Petersen, 2007;Zenk and Juenger, 2007;Sto¨ckigt et al, 2007;Ziegler and Facchini, 2008). Although it was argued earlier in the twentieth century that secondary metabolites arise spontaneously or with the aid of nonspecific enzymes, we have good evidence today that biosynthetic enzymes are highly specific in most instances and that most have been selected for this special task (although they often derive from common progenitors with a function in primary metabolism) (Wink, 2008b).…”