Catharanthus roseus enzyme

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.

Lee-Parson CWT, Shuler ML (2005) Sparge gas composition affects biomass and ajmalicine production from immobilized cell cultures of Catharanthus roseus. Enzyme MicrobTechnol 37 424-434. doi 10.1016/J.enzmictec.2005.02.016... 

Salutaridinol 7-0-acetyltransferase catalyzes the conversion of the phenanthrene alkaloid salutaridinol to salutaridinol-7-Oacetate, the immediate precursor of thebaine along the morphine biosynthetic pathway in P. somniferum (Fig. 10.7).26 Acetyl CoA-dependent acetyltransferases have an important role in plant alkaloid metabolism. They are involved in the synthesis of monoterpenoid indole alkaloids in medicinal plant species such as Rauwolfia serpentina. In this plant, the enzyme vinorine synthase transfers an acetyl group from acetyl CoA to 16-epi-vellosimine to form vinorine. This acetyl transfer is accompanied by a concomitant skeletal rearrangement from the sarpagan- to the ajmalan-type (reviewed in2). An acetyl CoA-dependent acetyltransferase also participates in vindoline biosynthesis in Catharanthus roseus, the source of the chemotherapeutic dimeric indole alkaloid vinblastine (reviewed in2). Acetyl CoA deacetylvindoline 4-O-acetyltransferase catalyzes the last step in vindoline biosynthesis. A cDNA encoding acetyl CoA deacetylvindoline 4-0-acetyltransferase was recently successfully isolated.27... 

Another cultured cell line of Catharanthus roseus (EU4A), which does not produce detectable amounts of vinblastine and other bisindole alkaloids, was also examined for its ability to transform 78 (183). Cell-free extracts of the culture line were prepared, and the 35,000 X g supernatant solution was used. Incubations with 2r-tritioanhydiovinblastine yielded a mixture from which radioactive vinblastine (52) was isolated. The labeled vinblastine was reisolated after unlabeled carrier was added and rigorously purified by successive thin-layer chromatography, reversed-phase HPLC, and crystallization to constant specific activity. Boiled extracts could not produce labeled 52, thus supporting the involvement of enzymes in the conversion process. 

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... 

Vincamine, vinblastine and vincristine are very important clinic alkaloids. They are produced naturally by plants vincamine by Vinca minor, and vinblascine and vincristine by Madagascar periwinkle Catharanthus roseus). The vindoline synthesis pathway starts with strictosidine and, via dehydrogeissoschizine, preakuammicine, stemmadenine and tabersonine, is converted to vindoline and vincristine (Figure 42). Conversion from vindoline to vinblastine is based on the NADH enzyme activity. Vinblastine and vincristine are very similar alkaloids. The difference is that vincristine has CHO connected to N, whereas vinblastine in the same situation has only CO3. This synthetic structural differences influence their activity. Vinblastine is used to treat Hodgkin s disease (a form of lymphoid cancer), while vincristine is used clinically in the treatment of children s leukaemia. Vincristine is more neurotoxic than vinblastine. 

Figure 75. The biochemical model for indole alkaloid formation in Catharanthus roseus. The arrows represent the direction of the formation and the flux of compounds in skeleton construction. On the diagram, enzymes are shown by a circle.

Kaltenbach, M. et al., Flavonoid hydroxylase from Catharanthus roseus. cDNA, heterologous expression, enzyme properties and cell-type specific expression in plants. Plant J., 19, 183, 1999. 

Geerlings A, Martinez-Lozano Ibanez M, Memelink J, van der Heijden R, Verpoorte R (2000) The strictosidine 6-D-glucosidase gene from Catharanthus roseus is regulated coordinately with other terpenoid-indole alkaloid biosynthetic genes and the encoded enzyme is located in the endoplasmic reticulum. J Biol Chem 275 3051-3056... 

Cathenamine (100) has been identified as an early intermediate in terpenoid indole alkaloid biosynthesis (cf. Vol. 8, p. 27). It has also been isolated from Guettarda eximia. Another alkaloid, 4,21-dehydrogeissoschizine (99), has now been isolated from this plant it is readily converted into (100) in alkaline solution.29 On incubation with an enzyme preparation from Catharanthus roseus cell cultures in the presence of NADPH at pH 7, (99) was converted into ajmalicine (102), 19-ep/-ajmalicine (103), and tetrahydroalstonine (104), which are the normal products with this enzyme preparation. In the absence of NADPH, cathenamine (100) accumulated.30 The reaction to give (100) proceeded linearly with time, and was dependent on the concentration of protein and substrate. No conversion occurred in the absence of enzyme. 

Within the natural products field, the investigation of complete biosynthetic pathways at the enzyme level has been especially successful for plant alkaloids of the monoterpenoid indole alkaloid family generated from the monoterpene gluco-side secologanin and decarboxylation product of tryptophan, tryptamine [3-5]. The most comprehensive enzymatic data are now available for the alkaloids ajmalicine (raubasine) from Catharanthus roseus, and for ajmaline from Indian Rauvolfia serpentina [6] the latter alkaloid with a six-membered ring system bearing nine chiral carbon atoms. Entymatic data exsist also for vindoline, the vincaleucoblastin (VLB) precursor for which some studies on enzymatic coupling of vindoline and catharanthine exist in order to get the so-called dimeric Catharanthus indole-alkaloids VLB or vincristine [7-9] with pronounced anti-cancer activity [10, 11]. 

Fig. 1 First example of a complete biosynthetic pathway to an alkaloid (ajmalicine = raubasine) based on enzymatic reactions elucidated by isolation of the single enzymes from Catharanthus roseus cell suspension cultures...

Terpenoid Indole Alkaloids.—Current knowledge on the biosynthesis of terpenoid indole alkaloids, with particular emphasis on the very important results obtained with enzyme preparations from tissue cultures of Catharanthus roseus, has been authoritatively reviewed.53 Further work on cell lines of C. roseus that are able to produce Aspidosperma-type alkaloids has been published54 (cf. Vol. 11, p. 19). 

In the transcript of a lecture, Zenk has reviewed58 the enzymic synthesis of ajmalicine and its biogenetic intermediates from secologanin and tryptamine in cell clones of Catharanthus roseus. 

Figure 7.9 Intercellular and subcellular trafficking in alkaloid biosynthesis. A. Tropane alkaloid biosynthesis in Hyoscyamus muticus. B. Terpenoid indole alkaloid biosynthesis in Catharanthus roseus. C. Trafficking of the berberine bridge enzyme in Papaver somniferum cell cultures.

IRMLER, S., SCHRODER, G., ST-PIERRE, B CROUCH, N.P., HOTZE, M., SCHMIDT, J., STRACK, D MATERN, U., SCHRODER, J Indole alkaloid biosynthesis in Catharanthus roseus new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase. Plant J., 2000,24, 797-804. 

GEERLINGS, A., MARTINEZ-LOZANO IBANEZ, M., MEMELINK, J., VAN DER HEIJDEN, R., VERPOORT, R., Molecular cloning and analysis of strictosidine P-D-glucosidase, an enzyme in terpenoid indole alkaloid biosynthesis in Catharanthus roseus. J. Biol. Chem., 2000,275,3051-3056. 

DE LUCA, V., BALSEVICH, J., TYLER, R.T., KURZ, W.G.W, Characterization of a novel V-methyltransferase (NMT) from Catharanthus roseus plants. Detection of NMT and other enzymes of the indole alkaloid biosynthetic pathway in different cell suspension culture systems. Plant Cell Rep., 1987,6,458-461. 

DE LUCA, V., FERNANDEZ, J.A., CAMPBELL, D., KURZ, W.G.W., Developmental regulation of enzymes of indole alkaloid biosynthesis in Catharanthus roseus. Plant Physiol, 1988, 86,447-450. 

MEIJER, A.H., CARDOSO, M.I.L., VOSKUILEN, J.T., DE WAAL, A., VERPOORTE, R., HOGE, J.H.C., Isolation and characterization of a cDNA clone from Catharanthus roseus encoding NADPH cytochrome P-450 reductase, an enzyme essential for reactions catalyzed by cytochrome P-450 mono-oxygenases in plants. Plant J., 1993,4,47-60. 

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.

The enzyme STR1 that was first characterized in Catharanthus roseus cell suspension cultures produces the central indole alkaloid intermediate H-3-a-(S)-strictosidine from tryptamine and secologanin (Fig. 8.9). It is well known that strictosidine represents the central intermediate precursor for several thousand indole alkaloids found in Nature. STR1 was the first gene to be cloned from R serpentina that involved a committed step in alkaloid biosynthesis.31 This was soon followed by the identification and isolation of an STR clone from Catharanthus roseus32 whose sequence was 80 % identical to the same gene from R serpentina.31... 

Tryptamine has been identified as a native compound in tomato,132 and the gene encoding tryptophan decarboxylase has been isolated from Catharanthus roseus,133 Plants grown on deuterium oxide incorporated more label into tryptamine than IAA, which was consistent with the result expected for a precursor of IAA. IAOx may be a YUCCA pathway intermediate for IAA biosynthesis in A. thaliana, and perhaps in rice and maize as well however, no enzyme has yet been identified for the conversion of A-hydroxyl tryptamine to IAOx. Because tryptamine is not a compound universally present in plants69 and deuterium oxide labeling ruled out tryptamine as an intermediate in tomato,132 the pathway would have to be species-specific. 

Figure 9.151 Determination of strictosidine synthetase activity by HPLC. Codeine (a), tryptamine (b), and strictosidine (c) were separated on a 4.0 (i.d.) X 250 mm LiChrosorb RP-8 Select B column at a flow rate of 1.0 mL/min. Incubation was for 30 minutes at 30°C with enzyme from Catharanthus roseus after ammonium sulfate precipitation (35-50% saturation) and gel filtration on Sephadex G-25, in the presence of 100 mM fi-D-gluconolactone. Injection volume was 8 pL and the UV detector was set at 0.02 AUFS. (From Pennings et al., 1989.)...

Grabowski, L., Heim, S., and Wagner, K.G., 1991, Rapid changes in the enzyme activities and metabolites of the phosphatidylinositol-cycle upon induction by growth substrates of auxin-starved suspension cultured Catharanthus roseus cells. Plant Sci. 75 33-38. 

Endo, T., Goodbody, A., Vukovic, J. and Misawa, M. (1986) Enzymes from Catharanthus roseus cell suspension cultures that couple vindoUne and catharanthine to form 3, 4 -anhydrovinblastine. Phytochemistry, 27,2147-9. 

Meijer, A.H., Verpoorte, R. and Hoge, J.H.C. (1993b) Regulation of enzymes and genes involved in terpenoid indole alkaloid biosynthesis in Catharanthus roseus.. Plant Res. (Special Issue), 3,145-64. 

Initially, the BR bios mthetic pathway was elucidated in Catharanthus roseus cell cultures by analysing the conversion products and intermediates (Fujioka et al, 1997). More recently, the biosynthesis of BRs has mainly been studied in Arabidopsis thaliam. Many of the genes encoding BR biosynthetic enzymes have been cloned using BR biosynthesis mutants of Arabidopsis thaliana, pea, tomato and rice. These mutants are BR deficient and revert to a wild-type phenotype following treatment with exogenous BRs. 

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