Alkaloids strictosidine synthase

Fig. 10.1 Reaction catalyzed by strictosidine synthase (Str) in monoterpenoid indole alkaloid formation.

The enzyme responsible for the stereospecific condensation of trypt-amine and secologanin 34) was called strictosidine synthase, and its presence was demonstrated by Treimer and Zenk 194) in a number of indole alkaloid-producing plants, including Amsonia salicifolia, Catharanthus roseus, Ochrosia elliptica, Rauwolfia vomitoria, Rhazya orientalis, Stem-madenia tomentosa. Vinca minor, and Voacanga africana. Enzyme activity as high as 1698 pkat/mg protein was observed for O. elliptica. No... 

Bracher, D. and Kutchan, T. M. 1992. Strictosidine synthase from Rauvolfia serpentina Analysis of a gene involved in indole alkaloid biosynthesis. Archives of Biochemistry and Biophysics, 294 717-723. 

Fig. 6 In vivo reprogramming of alkaloid biosynthesis in hairy roots of C. roseus by introduction of a mutant cDNA of the key enzyme strictosidine synthase (STR) with broader, unnatural substrate specificity leading to diversification of alkaloid content in roots following long-term feeding with 5-substituted tryptamines (X = Cl, Br, Me) [78]...

The power of engineered enzymes in the synthesis of novel alkaloids, to generate structural diversity and establish new alkaloid libraries, is best represented by the enzyme strictosidine synthase (STR1). 

CANEL, C., LOPES-CARDOSO, M.I., WH1TMER, S VAN DER FITS, L., PASQUALI, G., VAN DER HEIJDEN, R HOGE, J.H., VERPOORTE, R., Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus. Planta, 1998,205, 414-419. 

Fig. 8.1 Sequence of reactions and pathways involved in the biosynthesis of indole alkaloids in Catharanthus roseus. The dotted lines indicate multiple and/or uncharacterized enzyme steps. Tryptophan decarboxylase (TDC), Geraniol Hydroxylase (GH), Deoxyloganin synthase (DS), Secologanin Synthase (SLS) Strictosidine synthase (STR1), Strictosidine glucosidase (SG), Tabersonine-16-hydroxylase (T16H), Tabersonine 6,7-eposidase (T6,7E), Desacetoxyvindoline-4-hydroxylase (D4H), Deacetyl-vindoline-4-O-acetyltransferase (DAT) and Minovincinine-19-O-acetyltransferase (MAT) represent some of the enzyme steps that have been characterized.

Isolation of the stereospecific strictosidine s)mthase (STS) and formation of strictosidine with the 3a-(S) configuration proved conclusively that this was the natural precursor of the terpenoid indole alkaloids. Strictosidine occurs naturally in Rhazya stricta and the synthase has been isolated from a number of other species Amsonia salicifolia, A. tabemaemontam, Catharanthus pusillus, C. roseus, Rauvolfia verticillata, R. vomitoria, R. serpentina, Rhazya orientalis and Voacanga africana. The enzyme has been purified to homogeneity from R. serpentina (Hampp and Zenk, 1988). A comparison of the activity of STS from C. roseus roots, the only portion of the plant to contain ajmalicine, with that present in plant cell cultures producing the same alkaloid demonstrated that the plant cell cultures are far more metabolically active (Ziegler and Facchini, 2008). 

Figure 2.12 A hypothetical view of compartmentation of indole alkaloid biosynthesis in Catharanthus roseus. Enzymes located with dashed arrows are hypothetical and circles indicate membrane associated enzymes (after Meijer et at, 1 993b). Cl OH, geraniol-1 0-hydroxylase NMT, 5-adenosyl-L-methionine 11 -methoxy 2,16-dihydro-16-hydroxytabersonine N-methyltransferase DAT, acetylcoenzyme A deacetylvindoline 1 7-0-acetyltransferase OHT, 2-oxyglutarate-dependent dioxygenase SSpC, strictosidine-((3)-glucosidase SSS, strictosidine synthase.

Strictosidine synthase has a wide distribution among plants, although it is functionally expressed in a small group of taxa producing monoterpene indole alkaloids (see arrow in Fig. 7.17b). Related genes occur in animals and... 

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). 

Transcription factors that upregulate strictosidine synthase (132), as well as a transcription factor that coordinately upregu-lates expression of several terpenoid indole alkaloid biosynthetic genes, have been found (133). Several zinc finger proteins that act as transcriptional repressors to tryptophan decarboxylase and strictosidine synthase also have been identified (134). Manipulation of these transcription factors may allow tight control of the regulation of terpenoid indole alkaloid production. Interestingly, expression of a transcription factor from Arabidopsis thaliana in C. roseus cell cultures results in an increase in alkaloid production (135). 

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],... 

Fig. (3). Compartmentalization of the biosynthetic pathway of terpenoid indole alkaloids in plant cells. G10H geraniol 16-hydroxylase SLS secologanin synthase TDC tryptophan decarboxylase STR strictosidine synthase SGD strictosidine P-D-glucosidade T16H tabersonine 16-hydroxylase OMT S-adenosyl - L-methionine 16-hydroxytabereonine - 16-O-methyltransferase NMT S-adenosyl - /.-methionine 16-methoxy - 2,3-dihydro-3-hydroxytabersonine - A -methyltransferase D4H desacetoxy vindoline 4-hydroxylase DAT acetylcoenzyme A 4-O-deacetylvindoline 4-O-aeetyltransferase PRX peroxidase.

Fig. (4). Early steps of the biosynthesis of terpenoid indole alkaloids in Catharanthus roseus. Triple arrowheads indicate multiple steps. G10H geraniol 16-hydroxylase TDC tryptophan decarboxylase STR strictosidine synthase.

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. 

G. Pasquali, Regulation of the terpenoid indole alkaloid biosynthetic gene strictosidine synthase from Catharanthus roseus. Ph.D. Thesis, Leiden University, 1994. 

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... 

Biosynthesis The biosynthesis of these alkaloids begins with the key reaction, the condensation of tryptamine with secologanin to give strictosidine catalyzed by the enzyme strictosidine synthase. The biosynthesis was elucidated mainly by the use of plant cell suspension cultures. Cell cultures of Catharanthus and Rauvolfia species have been the subject of intense phytochemical studies. 

---