Báo cáo khoa học: Phenylalanine-independent biosynthesis of 1,3,5,8-tetrahydroxyxanthone A retrobiosynthetic NMR study with root cultures of Swertia chirata docx

Phenylalanine-independent biosynthesis of
1,3,5,8-tetrahydroxyxanthone
A retrobiosynthetic NMR study with root cultures of
Swertia chirata
Chang-Zeng Wang
1
, Ulrich H. Maier
1
, Michael Keil
2
, Meinhart H. Zenk
1
, Adelbert Bacher
3
,
Felix Rohdich
3
and Wolfgang Eisenreich
3
1
Biozentrum-Pharmazie, Universita
¨
t Halle, Halle/Saale, Germany;
2
Boehringer Ingelheim Pharma KG, Ingelheim, Germany;
3
Lehrstuhl fu
¨
r Organische Chemie und Biochemie, Technische Universita
¨
tMu

¨
nchen, Garching, Germany
Root cultures of Swertia chirata (Gentianaceae) were grown
with supplements of [1-
13
C]glucose, [U-
13
C
6
]glucose or
[carboxy-
13
C]shikimic acid. 1,3,5,8-Tetrahydroxyxanthone
was isolated and analysed by quantitative NMR analysis.
The observed isotopomer distribution shows that 1,3,5,8-
tetrahydroxyxanthone is biosynthesized via a polyketide-
type pathway. The starter unit, 3-hydroxybenzoyl-CoA, is
obtained from an early shikimate pathway intermediate.
Phenylalanine, cinnamic acid and benzoic acid were ruled
out as intermediates.
Keywords: hydroxybenzoate; isotope labelling; NMR
spectroscopy; retrobiosynthesis; xanthone.
The shikimate pathway (Fig. 1) supplies building blocks for
a wide variety of plant metabolites via phenylalanine (7),
such as lignin, stilbenes and flavonoids, or via early pathway
intermediates as specific precursors. Differentiation between
these alternatives is possible by retrobiosynthetic analysis
using general
13
C-labelled precursors (e.g. glucose; reviewed

in [1]). Using this approach, we previously showed that the
biosynthetic pathways of the tannic acid precursor, gallic
acid (8), and the bitter compound, amarogentin (9),
produced in Swertia chirata (Gentianaceae) branch off the
shikimate pathway at a level before phenylpyruvate (6)
(Fig. 1) [2,3].
We now exploit the retrobiosynthetic method to analyse
the biosynthesis of a xanthone derivative. Xanthones are
formed in at least 30 families of higher plants (e.g.
Gentianaceae and Guttiferae) [4,5]. 1,3,5,8-Tetrahydroxy-
xanthone (13, Fig. 2) is found in considerable amounts in
the roots of S. chirata. A root culture of the latter plant has
been used successfully in stable isotope incorporation
experiments aimed at analysing the biosynthesis of
amarogentin [3], and therefore this root culture appeared
to be well suited for the present study.
Early studies on the biosynthesis of xanthones suggested
that the aromatic ring A (Fig. 2) is assembled via a
polyketide-type pathway, whereas rings B and C are derived
from a C
6
–C
1
benzoic acid moiety in a similar way to
flavonoid biosynthesis [6]. More specifically, xanthones
were proposed to be biosynthesized from hydroxybenzoyl-
CoA and three molecules of malonyl-CoA (10)[7].An
enzyme catalysing the condensation of 3-hydroxybenzoyl-
CoA (11) and malonyl-CoA (10) to a benzophenone
intermediate (12, Fig. 2A) has been isolated from Hyperi-

cum androsaemum [8,9].
The biosynthetic origin of the hydroxybenzoyl-CoA
precursor (11) of xanthones is subject to controversy.
Thus,
14
C-labelled phenylalanine (7)wasreportedtobe
incorporated into xanthones from Gentiana lutea, albeit with
low incorporation rates [10], and label from cinnamic acid
and benzoic acid was diverted to the xanthone, mangostin, in
Garcinia mangostana [11,12]. Similarly, the benzoyl moiety of
xanthone was claimed to be derived from
L
-phenylalanine in
H. androsaemum via cinnamic acid, cinnamoyl-CoA, benz-
aldehyde, benzoic acid and hydroxybenzoic acid [13,14].
These reports supported earlier data on the origin of C
6
–C
1
units via the phenylpropanoid pathway [15–17]. On the other
hand,
14
C-labelled phenylalanine (7) and benzoic acid (15)
were not incorporated into xanthones in cell cultures of
Centaurium erythraea [13].
NMR experiments using
13
C-labelled samples of glucose
and shikimate described in this paper show unequivocally
that, in S. chirata, the xanthone precursor, 3-hydroxy-

benzoyl-CoA, is formed from an early shikimate pathway
intermediate (at a level before phenylpyruvate) and not
from phenylalanine (7) via cinnamic acid or benzoic acid.
Experimental procedures
Materials
[1-
13
C]Glucose and [U-
13
C
6
]glucose were from Omicron
(South Bend, IN, USA). [7-
13
C]Benzoic acid, [1-
13
C]
bromoacetic acid and
L
-[U-
13
C
9
]phenylalanine were from
Correspondence to W. Eisenreich, Lehrstuhl fu
¨
r Organische Chemie
und Biochemie, Technische Universita
¨
tMu

¨
nchen, Lichtenbergstr. 4,
D-85747 Garching, Germany. Fax: + 49 89 28913363,
E-mail:
Abbreviations: INADEQUATE, incredible natural abundance double
quantum transfer experiment.
(Received 14 March 2003, revised 30 April 2003,
accepted 14 May 2003)
Eur. J. Biochem. 270, 2950–2958 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03669.x
Cambridge Isotope Laboratories (Andover, MA, USA).
[U-
13
C
9
]Cinnamic acid was prepared by treatment of
L
-[U-
13
C
9
]phenylalanine with phenylalanine ammonia-lyase
in 0.5
M
sodium borate, pH 8.5. [carboxy-
13
C]Shikimic acid
was synthesized from
D
-mannose and [1-
13

C]bromoacetic
acid [18,19].
Incorporation experiments
Root cultures of S. chirata were grown with supplements of
[carboxy-
13
C]shikimate, [carboxy-
13
C]benzoate, [ring-
13
C
6
]-
cinnamic acid, [1-
13
C]glucose or a mixture of [U-
13
C
6
]glu-
cose and unlabelled glucose, as described previously [3].
Briefly, the cultures were grown in medium containing
glucose instead of sucrose as carbon source without
significant loss of viability or xanthone productivity. In the
first experiment, the cultures were supplemented with a
mixture of [1-
13
C]glucose and unlabelled glucose proffered
at a ratio of 1 : 2.3 (w/w). Although this experiment could
also have been performed with the labelled glucose as the

only carbon source, the labelled compound was diluted with
unlabelled glucose to reduce the cost. A second experiment
was performed using a mixture of [U-
13
C
6
]glucose and
unlabelled glucose at a ratio of 1 : 20 (w/w). In this case,
[U-
13
C
6
]glucose as the only carbon source would afford
uniformly
13
C-labelled products devoid of any biosynthetic
information; therefore, [U-
13
C
6
]glucose was diluted with
unlabelled glucose (1 : 20) giving products with specific
isotopomer compositions. The total concentration of glu-
cose was 167 m
M
in these experiments. In further experi-
ments, [carboxy-
13
C
1

]shikimate, [carboxy-
13
C]benzoate or
[ring-
13
C
6
]cinnamic acid were proffered at concentrations of
0.5 m
M
, respectively, in medium containing 30 g glucose per
litre. The cultures were incubated for 21 days.
Isolation of 1,3,5,8-tetrahydroxyxanthone
Plant material (fresh weight, 50 g) was pulverized under
liquid nitrogen. The cold slurry was transferred to a flask and
extracted three times with 200 mL methanol under a
nitrogen atmosphere for 15 min. The slurry was filtered.
The solution was concentrated to dryness under reduced
pressure. The residue (500 mg) was applied to a column of
silica gel (Silica Gel 60, 220–440 mesh, 20 · 1.8 cm; Merck,
Darmstadt, Germany), which was developed with a mixture
of chloroform and methanol (30 : 1, v/v). Fractions were
combined and concentrated to dryness under reduced
pressure. The residue was crystallised from methanol (yield,
30 mg).
Fig. 2. Polyketide-type biosynthesis of 1,3,5,8-tetrahydroxyxanthone
(13) with 3-hydroxybenzoyl-CoA (11) as starter unit.
Fig. 1. Shikimate pathway as the source of phenylalanine and other
plant metabolites. Equivalent positions originally derived from phos-
phoenolpyruvate (1) and erythrose 4-phosphate (2)areindicatedbyred

and green, respectively. The blue-coloured two-carbon fragment in
phenylpyruvate (6) and phenylalanine (7) is obtained from a phos-
phoenolpyruvate unit by decarboxylation of 5.
Ó FEBS 2003 Biosynthesis of 1,3,5,8-tetrahydroxyxanthone (Eur. J. Biochem. 270) 2951
Isolation of amino acids
In the experiments with
13
C-labelled glucose, the biomass,
after extraction of the xanthone derivative, was hydrolysed,
and phenylalanine was isolated as described previously [20].
The yield of phenylalanine isolated from 4 g root cells (dry
weight) was 5 mg.
NMR spectroscopy
1,3,5,8-Tetrahydroxyxanthone was dissolved in methanol-
D
4
and phenylalanine was dissolved in 0.1
M
DCl.
1
Hand
13
C NMR spectra were recorded at 500.13 MHz and
125.76 MHz, respectively, using a Bruker DRX500 spec-
trometer. The data were processed with standard Bruker
software (
XWINNMR
3.0). Two-dimensional incredible nat-
ural abundance double quantum transfer experiments
(INADEQUATE) [21] were performed with the Bruker

pulse program INAD using a 135 ° read pulse (11.5 ls).
Assessment of isotopomer composition
The relative abundance of
13
C at specific positions of a given
metabolite was calculated from the signal intensities in 1D
13
C NMR spectra (Fig. 3). Specifically, the signal integrals
were determined for each
13
C-NMR signal of a metabolite
from the labelling experiment and of the same compound at
natural
13
C abundance [22]. The ratios of the signal integrals
of the biolabelled compound and of the compound at
natural abundance were then calculated for each respective
carbon atom. Absolute
13
C abundances for certain carbon
atoms (i.e. for carbon atoms with at least one attached
hydrogen atom displaying a
1
H-NMR signal in a non-
crowded region of the spectrum) were then determined from
the
13
C coupling satellites in the
1
H-NMR spectra. As an

example, the relative fraction of the
13
C-coupled satellites
(J
CH
¼ 160 Hz; Table 1) in the global intensity of the
1
H-NMR signal for H6 of 1,3,5,8-tetrahydroxyxanthone
(13)(d ¼ 7.16 p.p.m.; Fig. 4) from the experiment with
[U-
13
C
6
]glucose accounted for 4.6% (Table 1). In other
Fig. 3.
13
C-NMR spectrum of 1,3,5,8-tetrahydroxyxanthone. (A)
Spectrum of a sample with natural
13
C abundance; (B) spectrum of a
sample from the experiment with [1-
13
C]glucose. Asterisks indicate
signals from impurities.
Table 1. NMR data of 1,3,5,8-tetrahydroxyxanthone samples isolated from root cultures of S. chirata.
Chemical shifts
(p.p.m.) Coupling constants (Hz)
Precursor
[U-
13

C
6
]Glucose [1-
13
C]Glucose
[carboxy-
13
C]-
Shikimate
d
13
C d
1
H J
HH
J
CC
a
J
CH
a
%
13
C
b
%
13
C
13
C

c
%
13
C
b
%
13
C
b
1 164.35 73.0(2), 62.8(8b) 4.6 28.9(2), 29.9(8b) 1.5 1.2
2 99.35 6.17(d) 2.2(4) 73.0(1), 66.3(3) 162(2) 4.2 31.1(1), 28.8(3) 6.2
d
1.1
3 168.05 66.8(4,2) 4.3 55.8(4, 2) 1.5 1.2
4a 159.36 74.7(4), 64.1(8b) 4.5 28.3(4), 31.2(8b) 1.4 1.2
4b 145.16 66.8(8a) 4.7 68.9(8a) 7.2 1.3
4 95.49 6.41(d) 2.2(2) 74.5(4a), 67.2(3) 168(4) 4.0 29.5(4a), 29.5(3) 6.0 1.0
5 138.33 69.0(6), 6.3(7) 4.3 16.8(6, 7) 1.9 1.2
6 124.68 7.16(d) 8.9(7) 69.2(5), 59(9) 160(6),
11(8)
4.6 15.5(5, 7),
45.2(7)
1.6 1.1
7 110.48 6.56(d) 8.8(6) 70.2(8), 59.5(6) 161(7) 4.7 11.2(8), 56.8(6, 8) 1.1 1.0
d
8 154.37 70.5(7) 4.3 65.8(7) 4.6 1.1
8a 108.71 67.0(4b), 55.5(9) 4.9 19.6(4b), 63.3(4b, 9) 1.2 1.1
8b 102.81 63.5(4a,1) 4.4 60.2(4a, 1) 5.5 1.1
9 185.80 55.7(8a) 4.5 56.4(8a) 1.5 14.3
a

Determined from spectra of
13
C-enriched samples. Coupling partners are given in parentheses.
b
Absolute
13
C abundance.
c
Calculated as
the fraction of
13
C coupled satellite pairs in the total signal intensity for a given carbon. Carbons coupled to the respective index carbon are
indicated in parentheses.
d
Determined from
13
C satellites in the
1
H-NMR signal of the index atom.
2952 C Z. Wang et al.(Eur. J. Biochem. 270) Ó FEBS 2003
words, 4.6% of 13 isolated from the experiment with
[U-
13
C
6
]glucose contained
13
C at position 6. The relative
13
C

abundances determined for all other positions in 13 were
then referred to this value, thus obtaining absolute
13
C
abundances for every carbon atom (%
13
CinTable1).
In NMR spectra of multiply labelled samples displaying
13
C
13
C couplings, each satellite signal in the
13
C-NMR
spectra was integrated separately. The relative fractions of
each respective satellite pair in the total
13
C-NMR signal
integral of a given carbon atom were then calculated
(%
13
C
13
C in Table 1). These values were normalized to the
13
C abundances. For example, the signal intensities of
the satellite signals reflecting [4b,8a-
13
C
2

]-13 (sample from
the experiment with [U-
13
C
6
]glucose) accounted for  20%
in the overall signal intensities for C-8a (Fig. 5 and %
13
C
13
C in Table 1). On the basis of the overall
13
C
abundance of C-8a (4.9%), the molar contribution of
[4b,8a-
13
C
2
]-13 was calculated as 1.0 mol% (see Fig. 7A).
Results
As a prerequisite for the interpretation of labelling data by
NMR spectrometry, unequivocal assignments of all signals
are required. Although
13
C NMR signal assignments of
1,3,5,8-tetrahydroxyxanthone (13) were published on the
basis of chemical-shift arguments [23], we independently
assigned all signals by 2D carbon-carbon correlation
experiments (INADEQUATE) (Fig. 6). This double-quan-
tum filtered experiment reveals scalar couplings between

adjacent
13
C atoms [21]; hence, only molecular species with
adjacent
13
Catomsinthesamemoleculeareobserved.
Because of the low abundance of doubly
13
C-labelled
molecules in natural
13
C abundance samples (i.e. with two
adjacent
13
C atoms in the same molecular species), the
experiment is inherently insensitive. However, this draw-
back can be overcome by the use of
13
C-enriched samples.
Therefore, we analysed, by INADEQUATE spectroscopy,
1,3,5,8-tetrahydroxyxanthone from the experiment with
[U-
13
C
6
]glucose which had acquired intact blocks of
13
C-labelled atoms from [U-
13
C

6
]glucose derived precursors
(see below). Ten pairs of carbon signals detected in the
spectrum provided a solid basis for the assignments
summarized in Table 1.
Some of the 1D
13
C-NMR signals of 1,3,5,8-tetra-
hydroxyxanthone from the experiment with [U-
13
C
6
]glucose
are displayed in Fig. 5. The signals show satellites attributed
to
13
C
13
C coupling involving one or two adjacent
13
C
atoms, which indicate that metabolic precursors carrying
two or more adjacent
13
C atoms have been incorporated
into the biosynthetic product.
Under the experimental conditions used, the proffered
[U-
13
C

6
]glucose is converted into a variety of multiply
13
C-
labelled intermediary metabolites such as carbohydrate
phosphates, pyruvate, and acetyl-CoA via the major glucose
utilization pathways (glycolysis and pentose phosphate
cycle). Simultaneously, intermediary metabolites are
Fig. 4.
1
H-NMR signal for H-6 of 1,3,5,8-tetrahydroxyxanthone from
the experiment with [U-
13
C
6
]glucose. Satellite signals due to
1
H
13
C
couplings are indicated.
Fig. 5.
13
C-NMR signals of 1,3,5,8-tetrahydroxyxanthone from the
experiment with [U-
13
C
6
]glucose. Satellite signals due to
13

C
13
Ccou-
plings are indicated.
Fig. 6. Part of an INAEDQUATE spectrum of 1,3,5,8-tetrahydroxy-
xanthone from the experiment with [U-
13
C
6
]glucose.
Ó FEBS 2003 Biosynthesis of 1,3,5,8-tetrahydroxyxanthone (Eur. J. Biochem. 270) 2953
produced with natural
13
C abundance from the natural
abundance glucose proffered in large excess.
Anabolic (biosynthetic) processes extract labelled and
unlabelled molecules at random from the intermediary
metabolite pools for utilization as building blocks. Natural
products biosynthesized under the experimental conditions
are therefore mosaics of labelled and unlabelled building
blocks. Hence, they represent complex mixtures comprising
a variety of
13
C-labelled isotopomers which can be present
at relatively high abundance compared with their occur-
rence in natural abundance material. A systematic decon-
volution of the multiplets in the
13
C-NMR spectrum gives
the molar fraction of each isotopomer that can be detected

within the sensitivity limits of NMR spectroscopy.
The multiply
13
C-labelled isotopomers of 1,3,4,8-tetra-
hydroxyxanthone detected in the sample from the
[U-
13
C
6
]glucose experiment are summarized in Fig. 7A.
Their relative abundance calculated from the fraction of
each satellite pair in the global intensity of each respective
signal (%
13
C
13
C in Table 1) can be referenced to the global
absolute
13
C abundance for each carbon atom. This
approach affords the molar fraction of each respective
isotopomer. For the subsequent discussion, it is convenient
to compress this information into a single icon (Fig. 7A) in
which the labelling patterns of each observed multiply
13
C-labelled isotopomer are indicated by lines connecting
the respective atom positions. The abundance in mol% is
indicated numerically for each observed isotopomer and is
also shown graphically by the relative width of the lines. The
dots in Fig. 7A represent isotopomers with a single enriched

carbon atom; the accompanying numbers indicate the
mol% excess of the respective [
13
C
1
]-isotopomer above the
natural
13
C abundance of 1.1%. A total of three single-
labelled and 11 multiply
13
C-labelled isotopomers showed
increased abundance compared with unlabelled material.
The symmetric labelling pattern of ring A in the
experiment with [U-
13
C
6
]glucose implies that the biosyn-
thetic pathway must involve a c
2
symmetric moiety which is
free to rotate before giving rise to ring A. This result is in full
accordance with the known polyketide-type pathway of
xanthone biosynthesis [7–9] via the benzophenone inter-
mediate 12 [8,9] comprising a c
2
symmetric trihydroxy-
phenyl moiety (Fig. 2).
A much simpler pattern of isotopomers was observed in

xanthone biosynthesized from a mixture of [1-
13
C
1
]glucose
and natural abundance glucose. Signals of 1,3,5,8-tetra-
hydroxyxanthone from the experiment with [1-
13
C]glucose
are shown in Fig. 3B. The corresponding signals from a
sample with natural
13
C abundance are shown for compar-
ison in Fig. 3A. It is obvious that five carbon atoms (C8,
C4b, C8b, C2 and C4) are significantly enriched in
13
C
above the natural abundance
13
C level. Quantitative ana-
lysis of the NMR signals (for details see Experimental
procedures) afforded absolute
13
C abundance values for all
carbon atoms of the target compound (Table 1, Fig. 7B).
The
13
C abundances at C8, C4b, C8b, C2 and C4 varied
between 4.6 and 7.2 mol%.
The isotopomer compositions for proteinogenic phenyl-

alanine from the experiments with [U-
13
C
6
]glucose and
[1-
13
C]glucose were determined as described above and are
shown in Fig. 7D,E, respectively. The structural formulas
have been oriented to match the labelling patterns of the
amino acid with those of ring C of the xanthone
derivative. In each experiment, it is immediately obvious
that the labelling pattern of the aromatic ring in phenyl-
alanine is closely similar to that of ring C in the xanthone
Fig. 8. Reconstruction of the labelling patterns of phosphoenolpyruvate
(1), erythrose 4-phosphate (2) and shikimate (3) from the observed
labelling patterns of phenylalanine (7). (A) From the experiment with
[1-
13
C]glucose. (B) From the experiment with [U-
13
C
6
]glucose. The
colours indicate equivalent positions biosynthetically derived from
phosphoenolpyruvate (in red), erythrose 4-phosphate (in green) or the
fragment of phosphoenolpyruvate after decarboxylation (in blue)
(Fig. 1).
Fig. 7. Labelling patterns of 1,3,5,8-tetrahydroxyxanthone (A–C) and
phenylalanine (D and E). (A), (D) From the experiment with

[U-
13
C
6
]glucose; bold lines connect
13
C-labelled carbon atoms that
were transferred from the same molecule of [U-
13
C
6
]glucose; filled dots
represent
13
C
1
-isotopomers with
13
C enrichments well above the nat-
ural abundance contributions; numbers indicate
13
C enrichments in
mol%.(B),(E)Fromtheexperimentwith[1-
13
C]glucose; filled dots
indicate carbon atoms that acquired significant
13
C label; the numbers
indicate
13

C abundances. (C) From the experiment with [carb-
oxy-
13
C]shikimate; the filled triangle indicates the carbon atom that
acquired significant
13
C label; the number indicates the
13
C abundance.
2954 C Z. Wang et al.(Eur. J. Biochem. 270) Ó FEBS 2003
derivative. It is also obvious that the similarity of the
labelling does not extend further to include the b carbon of
the tyrosine side chain and ring B of the tricyclic
compound. As discussed in more detail below, the
comparison between the isotopomer patterns shows con-
clusively that the specific precursor of 1,3,5,8-tetra-
hydroxyxanthone is a shikimate pathway intermediate
before the level of phenylpyruvate.
In line with that conclusion, the carboxylic group of
shikimate is incorporated into the xanthone derivative as
shown by the experiment with [carboxy-
13
C]shikimate.
1,3,5,8-[9-
13
C
1
]Tetrahydroxyxanthone was found with an
abundance of 14.3 mol% (Fig. 7C). No excess
13

C abun-
dance was observed in 1,3,5,8-tetrahydroxyxanthone from
experiments with [carboxy-
13
C]benzoate and [ring-
13
C
6
]cin-
namic acid as precursors (data not shown).
Discussion
The competing biosynthetic hypotheses proposed in the
literature for the biosynthesis of ring C of 1,3,5,8-tetra-
hydroxyxanthone can be subjected to a rigorous test by
prediction of the 1,3,5,8-tetrahydroxyxanthone labelling
pattern from the labelling patterns of central metabolic
intermediates. In the present case, it is sufficient to derive the
labelling patterns of erythrose 4-phosphate (2) and phos-
phoenolpyruvate (1) by dissection of the phenylalanine
labelling patterns according to the shikimate pathway, the
universal source of the carbon skeletons of aromatic acids.
The labelling patterns of phenylalanine (7) obtained by
hydrolysis of cell mass from the experiment with [1-
13
C]glu-
cose (Fig. 8A) or [U-
13
C
6
]glucose (Fig. 8B) are qualitatively

similar to those found with other plants [1,2]. The side chain
reflects the labelling pattern of phosphoenolpyruvate from
which it is biosynthetically obtained via the shikimate
pathway of aromatic amino-acid biosynthesis (Fig. 1). The
aromatic ring of phenylalanine reflects the labelling patterns
of C2–C3 of phosphoenolpyruvate and of C1–C4 of
erythrose 4-phosphate. Owing to the symmetry of the ring,
the ortho and meta carbon atoms become pairwise homo-
topic in the experiment with [1-
13
C]glucose. As a conse-
quence, only an averaged value of 5.4%
13
C can be obtained
for the ortho ring carbon atoms (reflecting C3 of phos-
phoenolpyruvate and C4 of erythrose 4-phosphate, respect-
ively). However, as the
13
C abundance for C3 of
phosphoenolpyruvate can be gleaned from the b-carbon
atom in the side chain (i.e. 5.8%
13
C), the enrichment for C4
of erythrose 4-phosphate can be determined as 5.0%
13
C
from the average value. The deduced labelling patterns of
the intermediary metabolites can then be used to reconstruct
the labelling patterns of shikimate (3) in each respective
experiment (Fig. 8A,B).

Fig. 9. Labelling pattern of the B and C ring of 1,3,5,8-tetrahydroxyxanthone (13) from the experiment with [1-
13
C]glucose. (A) Prediction via
phenylalanine (7), benzoic acid (14) and 3-hydroxybenzoic acid (15). (B) Prediction via an early shikimate pathway intermediate (e.g. 3); filled dots
indicate
13
C label; numbers indicate predicted
13
C abundances; the
13
C abundances of phenylalanine carbon atoms were determined experimentally
(see Fig. 7E); the
13
C composition of shikimate (3) was reconstructed from that of phenylalanine (7) by retrobiosynthetic analysis (see Fig. 8A).
(C) Experimentally determined. For other details, see also legends to Figs 7 and 8.
Ó FEBS 2003 Biosynthesis of 1,3,5,8-tetrahydroxyxanthone (Eur. J. Biochem. 270) 2955
The experimentally observed labelling patterns of phenyl-
alanine (7) and the reconstructed labelling patterns of
shikimate (3) were then used to predict hypothetical
labelling patterns of 1,3,5,8-tetrahydroxyxanthone (13)via
different biosynthetic routes. Specifically, Figs 9A and 10A
show the predictions of the labelling patterns of the
xanthone derivative via phenylalanine (7) and benzoic acid
(14). On the other hand, Figs 9B and 10B show the
prediction of the labelling pattern of the xanthone derivative
13 via an early shikimate intermediate (such as 3)actingas
the specific biosynthetic precursor. The predicted labelling
patterns via phenylalanine were at odds with the observed
isotopomer compositions in 13 (Figs 9C and 10C), whereas
the prediction via shikimate almost perfectly matched the

detected patterns. Therefore, the phenylalanine hypothesis
must be abandoned.
Hence, 1,3,5,8-tetrahydroxyxanthone biosynthesized in
S. chirata joins the growing list of secondary plant meta-
bolites that are derived from an early shikimate derivative as
opposed to a pathway via phenylalanine and cinnamate. A
hypothetical mechanism for the conversion of shikimic acid
(3) into 3-hydroxybenzoate (15) is shown in Fig. 11 [3]. We
propose that the vinylogous elimination of phosphate from
shikimic acid 3-phosphate (16) affords the dihydroxy-diene
intermediate 17 which appears to be well suited for a
subsequent dehydration yielding the aromatic ring system of
3-hydroxybenzoate (15). The CoA ester of 15 could then
provide the starter unit for the downstream steps of the
polyketide-type biosynthesis of 13.
In line with this interpretation, the experiment with
[carboxy-
13
C
1
]shikimate shows that the carboxylic group,
which is lost in the formation of phenylalanine and tyrosine
(Fig. 1) is in fact incorporated into ring B of 1,3,5,8-
tetrahydroxyxanthone (Figs 7C and 11). Phenylalanine,
tyrosine and metabolites downstream from the amino acids
would not have been advantageous to that labelling pattern.
By comparison with the [
13
C]glucose incorporation
studies, the data structure in the experiment with [

13
C]shiki-
mate is simple. This prompts the question whether the
incorporation experiments with [
13
C]glucoses and the meti-
culous deconvolution of the complex NMR spectra were
really necessary to solve the biosynthetic problem. The
answer lies in the difference between quantitative and
qualitative analysis. On closer examination, the shikimate
experiment shows only that at least a certain fraction of the
xanthone derivative was obtained from a shikimate inter-
mediate before the biosynthetic level of phenylpyruvate. On
the other hand, the comparison of the labelling patterns of
phenylalanine and the xanthone derivative from the experi-
ments with
13
C-labelled glucose shows, on the basis of
quantitative data, that the xanthone derivative is obtained
predominantly or exclusively from an early shikimate
derivative and not via phenylalanine. More specifically, in
the experiment with [U-
13
C
6
]glucose, several isotopomers of
Fig. 10. Labelling pattern of the B and C ring of 1,3,5,8-tetrahydroxyxanthone (13) from the experiment with [U-
13
C
6

]glucose. (A) Prediction via
phenylalanine (7), benzoic acid (14) and 3-hydroxybenzoic acid (15). (B) Prediction via an early shikimate pathway intermediate, such as shikimate
(3); bold lines connect
13
C-labelled carbon atoms that are transferred from the same molecule of [U-
13
C
6
]glucose; filled dots represent
13
C
1
isotopomers with
13
C enrichment significantly above the natural abundance contributions; numbers indicate
13
C enrichments in mol%; the
isotopomer composition of phenylalanine was determined experimentally (see Fig. 7D); the isotopomer composition of shikimate (3)was
reconstructed from that of phenylalanine (7) (see Fig. 8B). (C) Experimentally determined. For other details, see legend of Fig. 8.
2956 C Z. Wang et al.(Eur. J. Biochem. 270) Ó FEBS 2003
13 predicted to be synthesized by a route via phenylalanine
(7) could not be detected (for example [8a,8-
13
C
2
]-13 and
[4b,5,6-
13
C
3

]-13) (Fig. 10A,C). On the basis of the sensitivity
and the error limits of our NMR analysis, it can be
concluded that at least 98% of the biosynthetic xanthone
derivative was obtained from a shikimate pathway inter-
mediate before the biosynthetic level of phenylpyruvate.
This is confirmed independently by the experiment with
[1-
13
C]glucose. Thus, the retrobiosynthetic method affords
stringent, quantitative answers even in cases where a given
metabolite can be biosynthesized via more than one
metabolic pathway, which is by no means an esoteric or
farfetched problem. The recent history of terpene biosyn-
thesis research provides a striking example. Besides the well-
known mevalonate pathway, plants were recently shown to
operate a second pathway for the biosynthesis of isoprenoid
precursors via 1-deoxyxylulose 5-phosphate (for review, see
[24]). As a result of crosstalk between these pathways ([25]
and references cited therein), plant terpenes typically
comprise precursors from both pathways. Even though
the contribution of the mevalonate pathway to the vast
majority of the more than 20 000 plant terpenes is rather
small, incorporation experiments with isotope-labelled
mevalonate had been interpreted, incorrectly, in terms of a
universal mevalonate origin for all plant terpenes. Only
recently has retrobiosynthetic analysis shown that, in
contrast with this assumption, most plant terpenes are
obtained predominantly via the nonmevalonate pathway.
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft,the

Fonds der Chemischen Industrie and the Hans-Fischer-Gesellschaft.
The expert help of Angelika Werner and Fritz Wendling with the
preparation of the manuscript is gratefully acknowledged.
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