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(Last modification:
04. October 2009)
Benzophenone Synthase
(BPS)
(Liu et al., 2003;
Beerhues et al., 2007)
Benzophenone and Xanthone derivatives are widely present in Guttiferae, and a
recent review gives examples for compounds of considerable medical interest (Beerhues
et al., 2006). Very interesting is that benzophenone derivatives probably
play a role as pollinator attractants in flowers of
Hypericum calycinum
(Gronquist et al., 2001).
Early precursor feeding studies suggested that the backbone of the two
natural products is synthesized via a polyketide synthase reaction from
benzoyl-CoA or a derivative (see for example review:
Sultanbawa,
1980). In 1996, the first
demonstration of a benzophenone synthase (BPS) activity was published (Beerhues,
1996). This enzyme from
Centaurium
erythraea (Gentianaceae) clearly preferred 3-Hydroxybenzoyl-CoA against
Benzoyl-CoA, but work a bit later showed that this might be different in other
plants: The activity detected in Hypericum androsaemum preferred
Benzoyl-CoA (Schmidt and Beerhues, 1997).
In this case, the 3'-hydroxyl group present in
2,3',4,6-Tetrahydroxy-Benzophenone is introduced by the P450 enzyme Benzophenone
3'-Hydroxylase (Schmidt and Beerhues, 1997).
The further conversion to xanthones represents an
important branch point in different plants: In cultured cells of Centaurium
erythraea RAFN, 2,3',4,6-tetrahydroxybenzophenone (THBP) was shown to be
intramolecularly coupled to 1,3,5-trihydroxyxanthone,
whereas in cell cultures of Hypericum androsaemum L. it was coupled to
form the isomeric 1,3,7-trihydroxyxanthone.
In both cases the reactions are catalyzed by P450 activities (Peters
et al., 1998). The principles of the BPS reactions, the xanthone formation,
and the names of a few end products are shown in the figure below, and at least
the names of some natural products are given.
Fig. 1.
Benzophenone Synthase (BPS) and
Biosynthesis of Xanthones.
2,3',4,6-Tetrahydroxy-Benzophenone can be synthesized via different pathways:
either directly from 3-Hydroxybenzoyl-CoA (Centaurium erythraea),
or via P450-catalyzed hydroxylation of the 2,4,6-Trihydroxy-Benzophenone
synthesized from Benzoyl-CoA (Hypericum androsaemum). The oxidative ring
closure to the xanthones is catalyzed by P450 enzymes (Xanthone Synthase).
Benzophenone synthase (BPS)
was cloned from Hypericum androsaemum cell cultures, and simultaneously
also the chalcone synthase (CHS)
(Liu et al., 2003),
and this permitted some interesting mutagenesis studies on the molecular basis
of the functional differences. It should be noted that CHS did have some BPS
activity (about 22% of the activity with 4-coumaroyl-CoA), but that BPS was
completely inactive with the substrates typical for CHS (e.g. 4-coumaroyl-CoA).
As it turned out with CHS, three amino acids in CHS were the most interesting
with respect to the substrate preference; they were residues contributing to the
shape of the initiation/elongation cavity in the active site. Their exchanges
into BPS-type (Leu263 to Met, Phe265 to Tyr,
and Ser338 to Gly)
led to a strong reduction of the CHS-activity (about six-fold) and an increase
of the BPS activity by about 50%,
and thus to an enzyme that had a preference for benzoyl-CoA. Comparable
attempts to convert BPS into CHS failed: the mutants either retained the BPS
activity or had no activity at all (Liu et al., 2003).
One interesting question is the origin of the benzoyl residues used in the
biosynthesis, and here again there might be different strategies available:
either by degradation of cinnamoyl-CoA to benzoic acid (Hypericum androsaemum,
Hypericaceae) (Abd El-Mawla and Beerhues, 2002), or by
direct branching from the shikimate pathway to 3-hydroxybenzoic acid in
Centaurium erythraea (Gentianaceae) (Abd El-Mawla et
al., 2001) and in Swertia chirata (Gentianaceae) (Wang
et al., 2003). The principles are shown below.
Fig. 2.
Top:
Biosynthesis of benzoic acid from phenylalanine via cinnamoyl-CoA and
benzaldehyde.
Bottom: Biosynthesis of
3-hydroxybenzoic acid
directly from shikimic acid, via shikimic acid 3-phosphate.
Scheme modified from Abd El-Mawla and Beerhues (2002) and Wang et al. (2003).
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Update
27.09.2009
Conversion of BPS into Phenylpyprone
synthase
Citations:
Klundt et al. (2009), reviewed
in Beerhues and Liu (2009)
Recent work investigated the
biosynthetic potential of BPS, by directed mutagenesis of residues believed to
be important in formation of the active site pocket. A first series of
experiments aimed at conversion of BPS into CHS; the residues to be mutagenized
were picked from differences to the Medicago sativa CHS:
| |
Residue |
| Medicago sativa
CHS |
Leu214 |
Gly256 |
Phe265 |
Val196 |
Gly216 |
Leu263 |
Ser338 |
| Hypericum androsaemum
BPS |
Met217 |
Ala260 |
Tyr269 |
Met199 |
Ser219 |
Met267 |
Gly342 |
None of the mutants from BPS to CHS
residues turned out to be successful; none of the proteins had CHS activity.
Then the authors tested double and multiple mutants in these positions, but that
was also a failure: either the proteins were inactive or killed E. coli, or they
simply had the wild-type BPS activity.
The authors then proceeded to investigate a large
number of all sorts of mutations in these and other positions.
The results in almost all cases were pretty frustrating: with one exception,
they did not bring any useful insight. The only exception was, somewhat
surprisingly, a mutation of a residue that is actually conserved in both BPS
and CHS: Thr135 which corresponds to Thr132 in the CHS, a residue that has
been implicated in other experiments to be important for shaping the active site.
Thr135 was replaced by a large number of other amino acids, but
only one had a drastic effect; the change of Thr to Leu: it converted the BPS
into a phenylpyrone synthase, i.e. an enzyme carrying out with benzoyl-CoA only
two condensations, producing a phenylpyrone. Remarkably, Thr135 mutations into
Ser or Phe functionally resembled the wild-type (but with lower catalytic
activities), and the mutations to Gly, Ala,Val, Ile, Asp, and Tyr led to
inactive proteins! The authors then tried to rationalize the unexpected activity
of the Thr135 to Leu mutant by homology modelling. This suggested that the
mutation might open a new pocket that is smaller than in the wild-type, and thus limits the number of
condensations to two, rather than the three required for benzophenone
biosynthesis. The effect appeared to be unique to this particular amino acid.
This high specificity is the really interesting result of this study, and it
should be noted that it was not expected and not predicted. Among other things,
this highlights (again) that predictions from sequences and even modelling are
still risky.
Otherwise there is not much unusual or
novel. Pyrones from two condensations are typical derailment products of CHS
activities, at least in vitro (more...).
The phenylpyrone product of the BPS mutant has been identified as product of a
type III PKS with two condensation reactions already eleven years ago (Eckermann
et al., 1998), and that enzyme was the second type III PKS to be
crystallized and analyzed in much detail (more...).
And not to forget: such pyrone ring systems are often released as derailment
products in in vitro reactions, where a
polyketide product cannot be processed/folded into the correct final products,
as for example with the octaketide synthase proposed for anthrone biosynthesis
in Aloe (more...) and the hexaketide
synthase proposed for plumbagin biosynthesis (more...).
Actually, such ring-system is present in the natural product bisnoryangonin (a
styrylpyrone) that is proposed to be synthesized by a type III PKS (more...).
In this context it is interesting that phenylpyrones formed after only one
condensation had been identified as CHS-byproducts already many years ago (e.g.
Hrazdina et al., 1976):
in this case the diketide synthesized from a phenylpropanoyl-CoA ester is released and folded to a pyrone ring system (more...).
The principle is shown in Fig. 3 (below). However, it is important to note that
the pyrone backbones are not identical: the pyrone from the diketide lacks a
double bond present in the pyrone ring from a triketide (Fig. 3, compare A and
B). This is not trivial, because it makes biosynthesis predictions from natural
phenylpyrones more complicated: would it be possible to predict whether such
pyrones derive from benzoyl-CoA and two condensations (triketides) or from
phenylpropanoid-CoAs and one condensation (diketides)? That could be quite
difficult, considering that there might be modifications by tailoring reactions.
To my knowledge, there is only one phenylpyrone which has been investigated by
precursor feeding studies: Psilotonin in Psilotum nudum (a
primitive fern). In that case it seems quite clear that it is derived from a
diketide synthesized from 4-coumaroyl-CoA (Leete
et al., 1982). That case also illustrates the potential difficulties in
deducing biosynthetic routes from natural products: the lack of the 4-hydroxyl
group indicates that there must be a reducing step, but it is not known at which
stage this occurs. Such reductases are known or have been postulated in the
biosynthesis of several polyketide natural products, but the information on
these enzymes is scarce, except for the reductase in 6'-deoxychalcone
biosynthesis (more...).
Remarkably, several different type III PKS have been cloned from Psilotum
nudum, with some puzzling results: in addition to the expected CHS, there
was a protein clearly preferring isovaleryl-CoA, i.e. per definition a
valerophenone synthase (VPS); there were two cDNAs coding for proteins that
clearly had stilbene synthase (STS) activities; and one protein without
detectable activity. But there was no cDNA for a protein catalyzing a reaction
expected for psilotonin biosynthesis (Yamazaki
et al., 2001). This is really a bit strange, because stilbenes or
valerophenone derived products are not known from this plant. The most puzzling
was probably the protein without detectable activity, for various reasons (it
carried a replacement of a His in the catalytic triad, there was a Gln instead).
The reasons for not finding a cDNA for the postulated psilotonin synthase remain
obscure. To my mind it is also not really excluded that it is hiding in one of
the other enzymes, but not found because the reductase postulated for the
biosynthesis was absent: the absence of such tailoring reductases often pose
problems in finding the expected products in in vitro reactions (more...).
Fig. 3.
Biosynthetic
routes to phenyl-2-pyrones. See text for explanations.
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References
-
Klundt,
T., Bocola, M., Lütge, M., Beuerle, T., Liu, B., Beerhues, L., 2009. A
single amino acid substitution converts benzophenone synthase into
phenylpyrone synthase. Journal of Biological Chemistry 284, 30957-30964
Benzophenone metabolism provides a
number of plant natural products with fascinating chemical structures
and intriguing pharmacological activities. Formation of the carbon
skeleton of benzophenone derivatives from benzoyl-CoA and three
molecules of malonyl-CoA is catalyzed by benzophenone synthase (BPS), a
member of the superfamily of type III polyketide synthases. A point
mutation in the active site cavity (T135L) transformed BPS into a
functional phenylpyrone synthase (PPS). The dramatic change in both
substrate and product specificities of BPS was rationalized by homology
modeling. The mutation may open a new pocket that accommodates the
phenyl moiety of the triketide intermediate but limits polyketide
elongation to two reactions, resulting in phenylpyrone formation.
3-Hydroxybenzoyl-CoA is the second best starter molecule for BPS but a
poor substrate for PPS. The aryl moiety of the triketide intermediate
may be trapped in the new pocket by hydrogen bond formation with the
backbone, thereby acting as an inhibitor. PPS is a promising
biotechnological tool for manipulating benzoate-primed biosynthetic
pathways to produce novel compounds.
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Beerhues,
L., Liu, B., 2009. Biosynthesis of biphenyls and benzophenones - evolution
of benzoic acid-specific type III polyketide synthases in plants.
Phytochemistry 70, 1719-1727
Type III polyketide synthases (PKSs) generate a diverse array of
secondary metabolites by varying the starter substrate, the number of
condensation reactions, and the mechanism of ring closure. Among the
starter substrates used, benzoyl-CoA is a rare starter molecule.
Biphenyl synthase (BIS) and benzophenone synthase (BPS) catalyze the
formation of identical linear tetraketide intermediates from benzoyl-CoA
and three molecules of malonyl-CoA but use alternative intramolecular
cyclization reactions to form 3,5-dihydroxybiphenyl and
2,4,6-trihydroxybenzophenone, respectively. In a phylogenetic tree, BIS
and BPS group together closely, indicating that they arise from a
relatively recent functional diversification of a common ancestral gene.
The functionally diverse PKSs, which include BIS and BPS, and the
ubiquitously distributed chalcone synthases (CHSs) form separate
clusters, which originate from a gene duplication event prior to the
speciation of the angiosperms. BIS is the key enzyme of biphenyl
metabolism. Biphenyls and the related dibenzofurans are the phytoalexins
of the Maloideae. This subfamily of the Rosaceae includes a number of
economically important fruit trees, such as apple and pear. When
incubated with ortho-hydroxybenzoyl (salicoyl)-CoA, BIS catalyzes a
single decarboxylative condensation with malonyl-CoA to form
4-hydroxycoumarin. A well-known anticoagulant derivative of this
enzymatic product is dicoumarol. Elicitor-treated cell cultures of
Sorbus aucuparia also formed 4-hydroxycoumarin when fed with the
N-acetylcysteamine thioester of salicylic acid (salicoyl-NAC). BPS is
the key enzyme of benzophenone metabolism. Polyprenylated benzophenone
derivatives with bridged polycyclic skeletons are widely distributed in
the Clusiaceae (Guttiferae). Xanthones are regioselectively cyclized
benzophenone derivatives. BPS was converted into a functional
phenylpyrone synthase (PPS) by a single amino acid substitution in the
initiation/elongation cavity. The functional behavior of this Thr135Leu
mutant was rationalized by homology modeling. The intermediate triketide
may be redirected into a smaller pocket in the active site cavity,
resulting in phenylpyrone formation by lactonization.
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Liu, B., Falkenstein-Paul, H., Schmidt, W., Beerhues,
L., 2003.
Benzophenone synthase and chalcone synthase from Hypericum androsaemum
cell cultures: cDNA cloning, functional expression, and site-directed
mutagenesis of two polyketide synthases. Plant Journal 34, 847-855.
Benzophenone derivatives, such as polyprenylated benzoylphloroglucinols
and xanthones, are biologically active secondary metabolites. The
formation of their C13 skeleton is catalyzed by benzophenone
synthase (BPS; EC 2.3.1.151) that has been cloned from cell cultures of
Hypericum androsaemum. BPS is a novel member of the superfamily
of plant polyketide synthases (PKSs), also termed type III PKSs, with
53-63% amino acid sequence identity. Heterologously expressed BPS was a
homodimer with a subunit molecular mass of 42.8 kDa. Its preferred
starter substrate was benzoyl-CoA that was stepwise condensed with three
malonyl-CoAs to give 2,4,6-trihydroxybenzophenone. BPS did not accept
activated cinnamic acids as starter molecules. In contrast, recombinant
chalcone synthase (CHS; EC 2.3.1.74) from the same cell cultures
preferentially used 4-coumaroyl-CoA and also converted CoA esters of
benzoic acids. The enzyme shared 60.1% amino acid sequence identity with
BPS. In a phylogenetic tree, the two PKSs occurred in different clusters.
One cluster was formed by CHSs including the one from H. androsaemum.
BPS grouped together with the PKSs that functionally differ from CHS.
Site-directed mutagenesis of amino acids shaping the initiation/elongation
cavity of CHS yielded a triple mutant (L263M/F265Y/S338G) that preferred
benzoyl-CoA over 4-coumaroyl-CoA.
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Beerhues, L., Liu, B., Raeth, T., Klundt, T.,
Beuerle, T., Bocola, M., 2006.
Benzoic acid-specific
type III polyketide synthases. In: Rimando, A. M., Baerson, S. R. (Eds.), Polyketides: Biosynthesis, Biological Activities and Genetic Engineering,
American Chemical Society, Washington, D.C., pp. 97-108.
Benzophenone synthase (BPS) and biphenyl synthase (BIS) catalyze the
formation of the same linear tetraketide from benzoyl-CoA and three
molecules of malonyl-CoA. However, BPS cyclizes this intermediate via
intramolecular C6-C1 Claisen condensation, whereas BIS uses
intramolecular C2-C7 aldol condensation. Benzophenone derivatives
include polyprenylated polycyclic compounds with high pharmaceutical
potential. Biphenyl derivatives are the phytoalexins of the economically
important Maloideae.
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Eckermann, S., Schröder, G.,
Schmidt, J., Strack, D., Edrada, R.A., Helariutta, Y., Elomaa, P.,
Kotilainen, M., Kilpeläinen, I., Proksch, P., Teeri, T.H. and Schröder,
J.: New pathway to polyketides in plants. Nature (London) 396, 387-390
(1998).
The repertoire
of secondary metabolism (involving the production of compounds not
essential for growth) in the plant kingdom is enormous, but the genetic
and functional basis for this diversity is hard to analyse as many of
the biosynthetic enzymes are unknown. We have now identified a key
enzyme in the ornamental plant Gerbera hybrida (Asteraceae)
that participates in the biosynthesis of compounds that contribute to
insect and pathogen resistance. Plants transformed with an antisense
construct of
gchs2, a complementary DNA encoding a previously unknown function,
completely lack the pyrone derivatives gerberin and parasorboside. The
recombinant plant protein catalyses the principal reaction in the
biosynthesis of these derivatives: GCHS2 is a polyketide synthase that
uses acetyl-CoA and two condensation reactions with malonyl-CoA to form
the pyrone backbone of the natural products. The enzyme also accepts
benzoyl-CoA to synthesize the backbone of substances that have become of
interest as inhibitors of the HIV-1 protease. GCHS2 is related to
chalcone synthase (CHS) and its properties define a new class of
function in the protein superfamily. It appears that CHS-related enzymes
are involved in the biosynthesis of a much larger range of plant
products than was previously realized.
Request a reprint, more...
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Abd El-Mawla, A. M. A., Beerhues, L., 2002. Benzoic
acid biosynthesis in cell cultures of Hypericum androsaemum. Planta
214, 727-733.
Biosynthesis of benzoic acid from cinnamic acid has been studied in cell
cultures of Hypericum androsaemum L. The mechanism underlying
side-chain shortening is CoA-dependent and non-beta-oxidative. The
enzymes involved are cinnamate:CoA ligase, cinnamoyl-CoA hydratase/lyase
and benzaldehyde dehydrogenase. Cinnamate:CoA ligase was separated from
benzoate:CoA ligase and 4-coumarate:CoA ligase, which belong to xanthone
biosynthesis and general phenylpropanoid metabolism, respectively.
Cinnamoyl-CoA hydratase/lyase catalyzes hydration and cleavage of
cinnamoyl-CoA to benzaldehyde and acetyl-CoA. Benzaldehyde dehydrogenase
finally supplies benzoic acid. In cell cultures of H. androsaemum,
benzoic acid is a precursor of xanthones, which accumulate during cell
culture growth and after methyl jasmonate treatment. Both the
constitutive and the induced accumulations of xanthones were preceded by
increases in the activities of all benzoic acid biosynthetic enzymes.
Similar changes in activity were observed for phenylalanine
ammonia-lyase and the xanthone biosynthetic enzymes benzoate:CoA ligase
and benzophenone synthase.
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Abd El-Mawla,
A. M. A., Schmidt, W., Beerhues, L., 2001.
Cinnamic acid is a
precursor of benzoic acids in cell cultures of Hypericum androsaemum
L. but not in cell cultures of Centaurium erythraea RAFN. Planta
212, 288-293.
Benzoic acids are precursors of xanthone biosynthesis which has been
studied in cell cultures of Hypericum androsaemum (Hypericaceae)
and Centaurium erythraea (Gentianaceae). In both cell cultures,
methyl jasmonate induces the Intracellular accumulation of a new
xanthone. Under these inductive conditions, feeding experiments were
performed with [7-C-14]3-phenylalanine, [7-C-14]benzoic acid and
[7-C-14]3-hydroxybenzoic acid. All three precursors were efficiently
incorporated into the elicited xanthone in H. androsaemum,
whereas 3-hydroxybenzoic acid was the only precursor to be incorporated
into xanthones in C. erythraea. In addition, an appreciable
increase in phenylalanine ammonia-lyase activity occurred only in
methyl-jasmonate-treated cell cultures of H. androsaemum. Benzoic
acids thus appear to be formed by different pathways in the two cell
cultures studied. In H. androsaemum, benzoic acid is derived from
cinnamic acid by side-chain degradation. In C. erythraea
3-hydroxybenzoic acid appears to originate directly from the shikimate
pathway.
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Beerhues, L., 1996. Benzophenone synthase from
cultured cells of Centaurium erythraea.
FEBS Letters 383, 264-266.
A
central step in xanthone biosynthesis is the formation of the C-13
skeleton, i.e. an intermediate benzophenone. Biosynthesis of
2,3',4,6-tetrahydroxybenzophenone from m-hydroxybenzoyl-CoA and
malonyl-CoA was shown in cell-free extracts from cultured cells of
Centaurium erythraea. The enzyme catalyzing this reaction was named
benzophenone synthase.
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Gronquist, M., Bezzerides, A., Attygalle, A.,
Meinwald, J., Eisner, M., Eisner, T., 2001. Attractive and defensive
functions of the ultraviolet pigments of a flower (Hypericum calycinum).
Proceedings of the National Academy of Sciences of the United States of
America 98, 13745-13750.
The
flower of Hypericum calycinum, which appears uniformly yellow to
humans, bears a UV pattern, presumably visible to insects. Two
categories of pigments, flavonoids and dearomatized isoprenylated
phloroglucinols (DIPs), are responsible for the UV demarcations of this
flower. Flavonoids had been shown previously to function as floral UV
pigments, but DIPs had not been demonstrated to serve in that capacity.
We found the DIPs to be present in high concentration in the anthers and
ovarian wall of the flower, suggesting that the compounds also serve in
defense. Indeed, feeding tests done with one of the DIPs (hypercalin A)
showed the compound to be deterrent and toxic to a caterpillar (Utetheisa
ornatrix). The possibility that floral UV pigments fulfill both a
visual and a defensive function had not previously been contemplated.
DIPs may also serve for protection of female reproductive structures in
other plants, for example in hops (Humulus lupulus). The DIPs of
hops are put to human use as bitter flavoring agents and preservatives
in beer.
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Hrazdina, G., Kreuzaler,
F., Hahlbrock, K., Grisebach, H., 1976. Substrate
specificity of flavanone synthase from cell suspension cultures of parsley and
structure of release products in vitro. Archives of Biochemistry and
Biophysics 175, 392-399.
The substrate specificity of
an extensively purified flavanone synthase from light-induced cell
suspension cultures of Petroselinum hortense was investigated.
p-Coumaroyl-CoA was found to be the only efficient substrate for flavanone
synthesis, producing naringenin (5,7,4'-trihydroxyflavanone). Besides
4-hydroxy-6[4-hydroxystyryl]2-pyrone (F. Kreuzaler and K. Hahlbrock (1975)
Arch. Biochem. Biophys. 169, 84-90) two further release products of the
synthase reaction in vitro were identified as
4-hydroxy-5,6-dihydro-6(4-hydroxyphenyl)2-pyrone and p-hydroxybenzalacetone.
The apparent Km values for malonyl-CoA and p-coumaroyl-CoA in the reaction
leading to naringenin, and for p-coumaroyl-CoA in the reaction leading to
the styrylpyrone derivative were 35, 1.6, and 2.6 µM, respectively. With
caffeoyl-CoA as substrate only a very small amount of eriodictyol
(5,7,3',4'-tetrahydroxyflavanone) was formed besides relatively large
amounts of the corresponding styrylpyrone, dihydropyrone, and benzalacetone
derivatives. No flavanone formation was observed with feruloyl-CoA as
substrate, but again appreciable amounts of the three types of short-chain
release products were formed. No reaction at all took place with
cinnamoyl-CoA, p-methoxycinnamoyl-CoA, isoferuloyl-CoA, or
p-hydroxybenzoyl-CoA. None of the styrylpyrone, dihydropyrone, and
benzalacetone derivatives has been detected in the cell cultures in vivo.
The present results suggest that naringenin is the only natural product of
the synthase reaction and that further substitution in the B-ring of the
flavonoids occurs in parsley at or after the flavanone stage. The nature of
the smaller release products is consistent with the assumption of a stepwise
addition of acetate units from malonyl-CoA to the acyl moiety of the starter
molecule, p-coumaroyl-CoA.
Note:
The enzyme was first labelled as 'flavanone synthase', because the chalcone was so quickly converted in a non-enzymatic reaction to the flavanone that it was not detectable as the initial product. That was corrected a few years later with improved techniques
(see reference below), but the wrong name was used in the publications up to
that time:
Heller, W., Hahlbrock, K., 1980. Highly purified "flavanone synthase" from parsley catalyzes the formation of naringenin chalcone. Archives of Biochemistry and Biophysics 200, 617-619
(more...).
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Leete, E., Muir, A., Towers, G. H. N., 1982. Biosynthesis of
psilotin from [2',3'-13C2,1'-14C,4-3H]phenylalanine
studied with 13C-NMR. Tetrahedron Letters 23, 2635-2638.
The 6-phenyl-dihydro-a-pyrone moiety of psilotin is formed from
[2',3'-13C2,1'-14C,4-3H]phenylalanine
in the plant Psilotum nudum with retention of all the isotopes.
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Peters, S., Schmidt, W., Beerhues, L., 1998.
Regioselective
oxidative phenol coupling of 2,3',4,6-tetrahydroxybenzophenone in cell
cultures of Centaurium erythraea RAFN and Hypericum androsaemum
L. Planta 204, 64-69.
A
crucial step in plant xanthone biosynthesis is the cyclization of an
inter-mediate benzophenone to a xanthone. In cultured cells of Centaurium erythraea RAFN, 2,3',4,6-tetrahydroxybenzophenone (THBP)
was shown to be intramolecularly coupled to 1,3, 5-trihydroxyxanthone,
whereas in cell cultures of Hypericum androsaemum L. it was
coupled to form the isomeric 1,3,7- trihydroxyxanthone. These
regioselective cyclizations that occur ortho and para, respectively, to
the 3'-hydroxy group of the benzophenone depend on cytochrome P-450, as
shown by the effectiveness of established P-450 inhibitors and
blue-light- reversible carbon monoxide inhibition. Furthermore, the
reactions absolutely require NADPH and O2. The underlying reaction
mechanism is probably an oxidative phenol coupling that is catalyzed
regioselectively by xanthone synthases. These enzymes are proposed to be
cytochrome P450 oxidases. The intramolecular cyclizations of THBP to
1,3,5- and 1,3,7-trihydroxyxanthones catalyzed by the two xanthone
synthases represent an important branch point in the plant xanthone
biosynthetic pathway.
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Schmidt, W., Beerhues, L., 1997. Alternative
pathway of xanthone biosynthesis in cell cultures of Hypericum
androsaemum L. FEBS Letters 420, 143-146.
The
biosynthesis of xanthones was studied in cell cultures of Hypericum
androsaemum L. We have detected a new benzophenone synthase, for
which the preferred substrate is benzoyl-CoA, itself supplied by
3-hydroxybenzoate:coenzyme A ligase. The stepwise condensation of
benzoyl-CoA with three molecules of malonyl-CoA, catalyzed by
benzophenone synthase, yields 2,4,6- trihydroxybenzophenone. This
intermediate is subsequently converted by benzophenone 3'-hydroxylase, a
cytochrome P450 monooxygenase. These biosynthetic steps, leading to the
formation of 2,3',4,6-tetrahydroxybenzophenone, represent an alternative
pathway to that recently proposed for cell cultures of Centaurium
erythraea (Peters et al., Planta, 1998).
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Sultanbawa, M. U. S., 1980. Xanthonoids of
tropical plants.
Tetrahedron 36, 1465-1506.
No Abstract available.
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Wang, C.-Z., Maier, U. H., Keil, M., Zenk, M. H.,
Bacher, A., Rohdich, F., Eisenreich, W., 2003.
Phenylalanine-independent biosynthesis of 1,3,5,8-tetrahydroxyxanthone. A
retrobiosynthetic NMR study with root cultures of Swertia chirata.
European Journal of Biochemistry 270, 2950-2958.
Root
cultures of Swertia chirata (Gentianaceae) were grown with
supplements of [1-13C]glucose, [U-13C6]glucose or [carboxy-13C]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.
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- Yamazaki,
Y., Suh, D.-Y., Sitthithaworn, W., Ishiguro, K., Kobayashi, Y., Shibuya,
M., Ebizuka, Y., Sankawa, U., 2001. Diverse chalcone synthase
superfamily enzymes from the most primitive vascular plant, Psilotum
nudum. Planta 214, 75-84.
Psilotum nudum Griseb is a pteridophyte and
belongs to the single family (Psilotaceae) of the division, Psilophyta.
Being the only living species of a once populated division, P. nudum
is the most primitive vascular plant. Chalcone synthase (CHS; EC
2.3.1.74) superfamily enzymes are responsible for biosyntheses of
diverse secondary metabolites, including flavonoids and stilbenes. Using
a reverse transcription-polymerase chain reaction strategy, four
CHS-superfamily enzymes (PnJ, PnI, PnL and PnP) were cloned from P.
nudum, and heterologously expressed in Escherichia coli.
These four enzymes of 396-406 amino acids showed sequence identity of
>50% among themselves and to other higher-plant CHS-superfamily enzymes.
PnJ and PnP preferred p-coumaroyl-CoA and isovaleryl-CoA,
respectively, as starter CoA and catalyzed CHS-type ring formation,
indicating that they are CHS and phloriso valerophenone synthase,
respectively. On the other hand, PnI and PnL preferred cinnamoyl-CoA as
starter CoA and catalyzed stilbene synthase-type cyclization and thus
were determined to be pinosylvin synthases (EC 2.3.1.146). In addition,
PnE, which uniquely contains a glutamine in place of otherwise strictly
conserved histidine, had no apparent in vitro catalytic
activity. Phylogenetic analysis indicated that these P. nudum
clones form a separate cluster together with Equisetum arvense
CHS. This cluster of pteridophytes is located next to the cluster formed
by pine (gymnosperm) enzymes, in agreement with their evolutionary
relationships. Psilotum nudum represents a plant with the most
diverse CHS-superfamily enzymes and this ability to diverge may have
provided a survival edge during evolution.
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