Activity of the cytochrome P450 enzyme geraniol 10-hydroxylase and alkaloid production in plant cell cultures (original) (raw)
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Geraniol 10-hydroxylase, a cytochrome P450 enzyme involved in terpenoid indole alkaloid biosynthesis
FEBS letters, 2001
Geraniol 10-hydroxylase (G10H) is a cytochrome P450 monooxygenase involved in the biosynthesis of iridoid monoterpenoids and several classes of monoterpenoid alkaloids found in a diverse range of plant species. Catharanthus roseus (Madagascar periwinkle) contains monoterpenoid indole alkaloids, several of which are pharmaceutically important. Vinblastine and vincristine, for example, find widespread use as anticancer drugs. G10H is thought to play a key regulatory role in terpenoid indole alkaloid biosynthesis. We purified G10H from C. roseus cells. Using degenerate PCR primers based on amino acid sequence information we cloned the corresponding cDNA. The encoded CYP76B6 protein has G10H activity when expressed in C. roseus and yeast cells. The stress hormone methyljasmonate strongly induced G10h gene expression coordinately with other terpenoid indole alkaloid biosynthesis genes in a C. roseus cell culture. ß
FEBS Letters, 2001
Geraniol 10-hydroxylase (G10H) is a cytochrome P450 monooxygenase involved in the biosynthesis of iridoid monoterpenoids and several classes of monoterpenoid alkaloids found in a diverse range of plant species. Catharanthus roseus (Madagascar periwinkle) contains monoterpenoid indole alkaloids, several of which are pharmaceutically important. Vinblastine and vincristine, for example, find widespread use as anticancer drugs. G10H is thought to play a key regulatory role in terpenoid indole alkaloid biosynthesis. We purified G10H from C. roseus cells. Using degenerate PCR primers based on amino acid sequence information we cloned the corresponding cDNA. The encoded CYP76B6 protein has G10H activity when expressed in C. roseus and yeast cells. The stress hormone methyljasmonate strongly induced G10h gene expression coordinately with other terpenoid indole alkaloid biosynthesis genes in a C. roseus cell culture.
Phytochemical Analysis, 1999
The cytochrome P450 enzyme geraniol 10-hydroxylase plays an important role in the biosynthesis of pharmaceutically important alkaloids in Catharanthus roseus. An HPLC assay was developed for this enzyme based on the UV detection of the product 10-hydroxygeraniol after its separation from the substrate geraniol on a reversed-phase C-18 column. Furthermore, this system can be used for the UV detection of nerol, which is also a substrate for geraniol 10-hydroxylase, and its product 10-hydroxynerol. The presence of interfering enzymes which epoxidize rather than hydroxylate geraniol and nerol could also be detected. The developed HPLC assay was validated with respect to the incubation time (linear up to 45 min) and the amount of protein added per incubation (linear up to 400 mg of protein). The HPLC assay will be a useful tool during the purification of geraniol 10-hydroxylase from cell suspension cultures of Catharanthus roseus.
Plant Physiology and Biochemistry, 1999
The cytochrome P450 enzyme geraniol IO-hydroxylase plays an important role in the biosynthesis of terpenoid indole alkaloids in suspension cultures of Cutharnnthus roseus. The activity of this enzyme was induced by the treatment of cells with phenobarbital, and inhibited by treatment with ketoconazole. The alkaloid accumulation increased after phenobarbital treatment whereas it decreased after ketoconazole treatment. Phenobarbital and ketoconazole did not affect the in vivo conversion rate of loganin to secologanin, a reaction proposed to be catalyzed by a cytochrome P450 enzyme. 0 Elsevier, Paris Alkaloids / Catharanthus roseus I cytochrome P450 I geraniol lo-bydroxylase I iridoids I ketoconazole I phenobarbital DMSO, dimetbylsulphoxide / DW, dry weight / EDTA, ethylenedinitrilo tetraacetic acid / FW, fresh weight / GlOH, geraniol lo-hydroxylase I NAA, naphthalene acetic acid
Journal of Agricultural and Food Chemistry, 2011
Geraniol 10-hydroxylase (G10H), a cytochrome P450 monooxygenase, has been reported to be involved in the biosynthesis of terpenoid indole alkaloids. The gene for Catharanthus roseus G10H (CrG10H) was cloned and heterologously expressed in baculovirus-infected insect cells. A number of substrates were subjected to assay the enzyme activity of CrG10H. As reported in a previous study, CrG10H hydroxylated the monoterpenoid geraniol at the C-10 position to generate 10hydroxygeraniol. Interestingly, CrG10H also catalyzed 3 0-hydroxylation of naringenin to produce eriodictyol. Coexpression of an Arabidopsis NADPH P450 reductase substantially increased the ability of CrG10H to hydroxylate naringenin. The catalytic activity of CrG10H was approximately 10 times more efficient with geraniol than with naringenin, judged by the k cat /K m values. Thus, G10H also plays an important role in the biosynthetic pathway of flavonoids, in addition to its previously described role in the metabolism of terpenoids.
Journal of Plant Physiology - J PLANT PHYSIOL, 2005
Geraniol 10-hydroxylase (G10H) is a P450 containing enzyme which is the first committed step in the biosynthesis of monoterpene indole alkaloids (MIAs), including the Catharanthus roseus-anticancer drugs vinblastine and vincristine. It is thought that G10H has a regulatory role in MIA production. In the present paper, we report the characterization of a polyclonal serum raised against the purified G10H polypeptide. Anti-G10H IgG was able to inhibit the G10H activity and also recognized the G10H polypeptide from C. roseus and other plants producing MIAs. These results establish the usefulness of this antiserum as a biochemical tool for the study of G10H regulation.
Effect of culture process on alkaloid production by Catharanthus roseus cells
Journal of Biotechnology, 1991
The processes for production of indole alkaloids in shake flask suspension cultures of Catharanthus roseus cells using Zenk's alkaloid production medium (APM) were evaluated. The 1-stage process consisted of inoculating APM and incubating for 15 days. The 2-stage process involved 6 d of cultivation in growth medium followed by 15 d of incubation in APM. Growth, main nutrient consumption and alkaloid production were monitored. Both culture processes produced ~ 20 g dw per I of biomass. However, 2-stage cultures yielded an inorganic nutrient richer and more active plant cell biomass, richer in inorganic nutrients, as indicated by higher (> 70%) nutrient availability and consumption. Total and individual indole alkaloid production were 10 times higher (740 mg 1-~ and 25 to 4000 /xg per g dw, respectively) for 2-stage than for 1-stage cultures. For both processes, highest alkaloid productivity coincided with complete extracellular consumption of major inorganic nutrients, especially nitrate, by the cells. Complete carbohydrate consumption in 2-stage cultures resulted in a 40% decline in produc-Correspondence to: J. Archambault, Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Ave., Montreal, Canada H4P 2R2. Abbreviations: 2,4 D: 2,4 dichlorophenoxyacetic acid; APM: alkaloid production medium; AB5:B5 growth medium with IAA; B5: plant cell basal growth medium; KLa: mass transfer coefficient; TIA: total indole alkaloid content; SM: secondary metabolites. 2 tion. Small but significant (~ 10%) product release was observed for both culture regimes, which seemed not to be related to cell lysis.
Catharanthus terpenoid indole alkaloids: biosynthesis and regulation
Phytochemistry Reviews, 2007
Catharanthus roseus is still the only source for the powerful antitumour drugs vinblastine and vincristine. Some other pharmaceutical compounds from this plant, ajmalicine and serpentine are also of economical importance. Although C. roseus has been studied extensively and was subject of numerous publications, a full characterization of its alkaloid pathway is not yet achieved. Here we review some of the recent work done on this plant. Most of the work focussed on early steps of the pathway, particularly the discovery of the 2-C-methyl-d-erythritol 4-phosphate (MEP)-pathway leading to terpenoids. Both mevalonate and MEP pathways are utilized by plants with apparent cross-talk between them across different compartments. Many genes of the early steps in Catharanthus alkaloid pathway have been cloned and overexpressed to improve the biosynthesis. Research on the late steps in the pathway resulted in cloning of several genes. Enzymes and genes involved in indole alkaloid biosynthesis and various aspects of their localization and regulation are discussed. Much progress has been made at alkaloid regulatory level. Feeding precursors, growth regulators treatments and metabolic engineering are good tools to increase productivity of terpenoid indole alkaloids. But still our knowledge of the late steps in the Catharanthus alkaloid pathway and the genes involved is limited.
Phytochemistry, 1995
The possible limitation of the rate of biosynthesis of terpenoid indole alkaloids by low enzyme levels was investigated in two cell lines of Tabernaemontana divaricata with different terpenoid indole alkaloid biosynthetic capacities. The activities of tryptophan decarboxylase (TDC), strictosidine synthase (SSS), strictosidine glucosidase (SG), isopentenyl pyrophosphate isomerase (IPP isomerase) and geraniol 10-hydroxylase (G10H) of both cell lines were compared. The activities of TDC, SSS and IPP isomerase did not show a direct relationship with the biosynthetic capacity but SG and GIOH might be limiting. In order to test whether the availability of the terpenoid precursor limits the biosynthesis of the terpenoid indole alkaloids, Ioganin was fed to the cultures. Loganin-feeding did not influence any of the measured enzyme activities but increased the terpenoid indole alkaloid accumulation of both cell lines to similar levels. A five-fold increase was observed for the accumulating line and a more than 100-fold increase for the low-accumulating one. Strictosidine accumulated mainly in the low-accumulating cell line which has high TDC and low SG activity; the amounts and types of the other terpenoid indole alkaloids which accumulated were similar in both lines. From this it can be concluded that the biosynthesis of terpenoid indole alkaloids in both cultures is limited by the availability of terpenoid precursors; this pathway is not saturated with substrates under normal culture conditions.