Antitumor Agents: Taxol and Taxanes – Production by Yew Cell Culture
425
of an oxetane ring system, substitution
pattern, and the ester side chains. Studies
on Taxol
biosynthesis have been con-
ducted particularly by the groups of R.
Croteau and H. Floss in the United States.
Considerable advances have been made
both on the biochemical and molecular
aspects of taxane biosynthesis. As diter-
penes, the taxane skeleton has been shown
to be derived from isopentenyl diphos-
phate (IPP), synthesized via the 1-deoxy-
D-xylulose-5-phosphate (DXPS) pathway,
on the basis of
13
C labeled glucose stud-
ies. This plastidic pathway functions in
parallel with the classic mevalonate de-
pendent route, which is now believed
to be responsible for the assembly of
IPP for the synthesis of sesquiterpenes,
triterpenes, and phytosterols. However,
the mevalonate pathway may be at least
partly involved in taxane assembly. Virtu-
ally, all productive cultures of yew species
are grown in dark (see below), indicating
that the DXPS pathway is likely operat-
ing in proplastids or poorly differentiated
chloroplasts, a scenario that is reinforced
by the well established suppression of
chloroplast differentiation and photosyn-
thetic pathways by high sucrose levels used
in culture media. The ±rst committed step
in the assembly of Taxol
and related
taxanes is the cyclization of the univer-
sal diterpenoid precursor geranylgeranyl
diphosphate to taxa-4-(5), 11(12)-diene, a
slow, but apparently not rate-limiting re-
action, catalyzed by a diterpene cyclase
(Fig. 3).
Cloning of the cDNA of taxadiene syn-
thase in
T. brevifolia
by a homology-based
PCR strategy revealed a 79-KDa peptide
size and an N-terminal plastid transit pep-
tide, in agreement with the plastid location
of the diterpene biosynthesis. Heterolo-
gous expression of taxadiene synthase in
bacteria followed by kinetic studies showed
that, unlike several terpene synthases in
plants, taxadiene synthase produces essen-
tially one product and not multiple taxadi-
ene isomers. The second speci±c step in
taxane assembly involves the cytochrome
P450-dependent hydroxylation at the C-5
position of the taxane skeleton, with allylic
rearrangement, yielding taxa-4(20),11(12)-
diene-5
α
-ol. This intermediate is acetylated
at the hydroxyl in C-5 by an acetyl trans-
ferase to yield taxa-4(20),11(12)-dien-5
α
-yl
acetate (Fig. 3). This is likely the third
speci±c step in Taxol
assembly and is
responsible for generating the 4(20)-en-5
α
-
acetoxy functional grouping from which
the oxetane D-ring is established. A P450-
dependent monooxygenase catalyzes the
hydroxylation of the later molecule, result-
ing
in
taxa-4(20),11(12)-dien-5
α
-acetoxy-
10
β
-ol. An additional cytochrome P450
cDNA clone encoding a product capable
of hydroxylating taxadien-5
α
-ol at the C-13
position has also been identi±ed.
The exact order of hydroxylation steps
and the sequence of intermediates in
the Taxol
biosynthetic pathway are not
clearly established, and it may be pos-
sible that various interconnected routes
to this compound exist. The same ap-
plies to acylation reactions and the ex-
act
timing
of
epoxidation.
After
oxy-
genation, various hydroxylated positions
in the taxane skeleton undergo esteri±-
cation.
These acylation reactions appear to span
early and later steps in the pathway.
Whereas acetylation of the C-5 hydroxyl
group occurs early in biosynthesis, acetyla-
tion at the C-10 hydroxyl and benzoylation
at the C-2 hydroxyl take place later. Clones
of cDNAs encoding these three taxane
o
-acyltransferases have been identi±ed.
The oxetane ring could arise by epoxi-
dation of the 4(20)-double bond of the
α
-acetoxy group, followed by migration of
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