Strictosidine synthase from secologanin

All terpenoid indole alkaloids are derived from tryptophan and the iridoid terpene secologanin (Fig. 2b). Tryptophan decarboxylase, a pyridoxal-dependent enzyme, converts tryptophan to tryptamine (62, 63). The enzyme strictosidine synthase catalyzes a stereoselective Pictet-Spengler condensation between tryptamine and secologanin to yield strictosidine. Strictosidine synthase (64) has been cloned from the plants C. roseus (65), Rauwolfla serpentine (66), and, recently, Ophiorrhiza pumila (67). A crystal structure of strictosidine synthase from R. serpentina has been reported (68, 69), and the substrate specificity of the enzyme can be modulated (70). 

Strictosidine is produced, stereospecifically, from tryptamine and secologanin by strictosidine synthase, isolated from several species producing monoterpene indole alkaloids. The enzyme was cloned and can be expressed in large quantity (Fig. 37). 

The basic stmcture of monoterpenoid indole alkaloids includes an indole nucleus derived from tryptophan via tryptamine (L) and a versatile C9 or CIO unit arising from the monoterpenoid secologanin (LI). Strictosidine synthase catalyzes the synthesis of strictosidine (LII) from tryptamine and secologanin (Scheme XXIV) [76],... 

The stereospecific condensation of secologanin and tryptamine is catalyzed by the enzyme strictosidine synthase (SSS, EC 4.3.3.2). Smith 193) postulated that strictosidine is the key intermediate for the terpenoid indole alkaloids. StOckigt and Zenk (194,195) and Scott etal. (196) showed that this intermediate indeed is formed through a specific enzyme, and its presence in a series of plants producing terpenoid indole alkaloids was shown (197). The first partial purification of SSS from C. roseus was reported by Treimer and Zenk (198) and by Mizukami et al. (199). The molecular weight was estimated to be between 34 and 38 kDa. The enzyme is soluble and does not require any cofactors. 

Based on knowledge of a biosynthetic pathway one can select certain steps which could be of interest for bioconversion of (a) readily available precursor(s). This could, for example, be stereospecific reactions, like the reduction of quinidinone in quinine or quinidine and the epoxidation of atropine to scopolamine. For the bioconversion one can consider using plant cells [e.g., the production of L-dopa from tyrosine by immobilized cells of Mucuna pruriens (10)] or isolated enzymes from the plant itself. An interesting example of the latter is the (5)-tetrahydroprotoberberine oxidase (STOX) enzyme, which oxidizes (5)-reticuline but not its stereoisomer (11). This feature can be used in the production of (i )-reticuline from a racemic mixture (see below). Immobilized strictosidine synthase has been successfully used to couple secologanin and tryptamine. The gene for this enzyme has been isolated from Rauvolfia (6) and cloned in Escherichia coli, in which it is expressed, resulting in the biosynthesis of active enzyme (7). The cultured bacteria produced 20 times more enzyme... 

The enzyme strictosidine synthase (EC 4.3.3.2) is responsible for the stereospecific coupling of tryptamine and secologanin, yielding strictosidine (Fig. 12). This glucoalkaloid is the precursor for all terpenoid indole and related alkaloids, including among others the Cinchona quinoline alkaloids. Hampp and Zenk (707) isolated and purified this enzyme to homogeneity from a cell suspension culture of R. serpentina. The enzyme could successfully be immobilized on CNBr-activated Sepharose 4B, as was reported for this enzyme isolated from Catharanthus roseus (102,708). It proved to be more stable than the C. roseus enzyme the half-life of the immobilized enzyme was 100 days at a temperature of 37°C. 

A key step in indole alkaloid biosynthesis is the formation of strictosidine from tryptamine and the aldehyde secologanin [357, 358]. This reactimi is catalyzed by the enzyme strictosidine synthase. The crystal structure of the enzyme has been determined and the binding site identified [359]. Site-directed mutagenesis has been used to identify both the active site amino acids and to modify the substrate specificity of the enzyme [360]. The enzymatic mechanism has been compared with the H -catalyzed reaction in solution and they appear to be similar, based on... 

When tryptamine and secologanin were incubated with an enzyme preparation from a Catharanthus cell culture in the presence of a 3-glucosidase inhibitor (D-t/-gluconolac-tone), only strictosidine (3) was formed. The reaction clearly was enzyme dependent. The enzyme, strictosidine synthase, a single polypeptide with 30,000 MW (Hampp and Zenk, 1988 Stockigt and Zenk, 1977), is found in the plant vacu-... 

Hallard D et al (1997) Suspension cultured transgenic cells of Nicotiana tabacum expressing tryptophan decarboxylase and strictosidine synthase cDNAs from Catharanthus roseus produce strictosidine upon feeding of secologanin. Plant Cell Rep 17 50-54... 

All monoterpenoid indole alkaloids are synthesized from strictosidine 44, which is a general biosynthetic intermediate formed by enzymatic condensation of trypt-amine with secologanin [162]. Recently, cDNA coding strictosidine synthase (E.C. 4.3.3.2) was isolated from 0. pumila and expressed further in E. coli. The enzyme has been characterized regarding its substrate specificity toward a number of tryptamines and activity regulation [163]. In another work distribution of strictosidine activity in O. pumila, tissues were shown to correlate with the pattern of respective mRNA expression [164]. 

Gene transfer from the angiosperm Catharanthus roseus, and over-expression in the bacterium Escherichia coli, yielded the synthase for strictosidine, a known alkaloid of the tryptophan-secologanin class (Scott 1992). A similar strategy has clarifted the biosynthesis of hydrogenobyrinic acid, an advanced precursor of vitamin B,2 (Scott 1994). 

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