ALKALOIDS - Natural Products in Chemical Biology - page 214

hybridization studies (109). However, additional studies have suggested that these
genes also are observed in the epidermis and laticifers (110). Studies of the local-
ization of vindoline biosynthetic enzymes by using immunocytochemistry and
in situ RNA hybridization strongly suggest that the mid-part of the vindoline
pathway (tryptophan decarboxylase, strictosidine synthase, and tabersonine-16-
hydroxylase) takes place in epidermal cells of leaves and stems. However, the
later steps catalyzed by desacetylvindoline 4-hydroxylase and desacetylvindoline
O-acetyltransferase take place in specialized cells, the laticifers, and idioblasts
(109-112). As with isoquinoline alkaloid biosynthesis, deconvolution of the
enzyme localization patterns remains a challenging endeavor.
9.3.5 Vinblastine
Vinblastine is a highly effective anticancer agent currently used clinically against
leukemia, Hodgkin's lymphoma, and other cancers. (113, 114). Vinblastine is
derived from dimerization of vindoline and another terpenoid indole alkaloid,
catharanthine. The dimerization of catharanthine and vindoline is believed to pro-
ceed via the formation of an iminium intermediate with catharanthine (Fig. 9.2e).
This iminium intermediate is reduced to form anhydrovinblastine, a naturally
occurring compound in C. roseus plants (115). In support of this mechanism,
anhydrovinblastine is incorporated into vinblastine and vincristine in feeding
studies (116-119).
Peroxidase containing fractions of plant extracts were found to catalyze the
formation of the bisindole dehydrovinblastine from catharanthine and vindoline
(120, 121). A peroxidase from C. roseus leaves has been demonstrated to convert
vindoline and catharanthine to anhydrovinblastine in vitro (122, 123). Because
the dimerization of these C. roseus alkaloids also can be catalyzed by peroxidase
from horseradish in reasonable yields (124), it is interesting to speculate that
anhydrovinblastine may be a by-product of isolation; after lysis of the plant
material, nonspecific peroxidases are released from the vacuole and may act on
vindoline and catharanthine.
9.3.6 Metabolic Engineering of Terpenoid Indole Alkaloids
Strictosidine synthase and tryptophan decarboxylase have been overexpressed
in C. roseus cell cultures (125, 126). Generally, overexpression of tryptophan
decarboxylase does not seem to have a significant impact on alkaloid production,
although overexpression of strictosidine synthase does seem to improve alkaloid
yields. Overexpression of tryptophan and secologanin biosynthetic enzymes in
C. roseus hairy root cultures resulted in modest increases in terpenoid indole alka-
loid production (127, 128). Secologanin biosynthesis seems to be the rate-limiting
factor in alkaloid production (129). Precursor-directed biosynthesis experiments
with a variety of tryptamine analogs suggest that the biosynthetic pathway can be
used to produce alkaloid derivatives (130). Strictosidine synthase and strictosi-
dine glucosidase enzymes also have been expressed successfully heterologously