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(Last
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Seven
condensation reactions:
Octaketide Synthase (OKS) in Hypericin Biosynthesis
in Hypericum perforatum
Hypericum perforatum
(St. John's Wort; Echtes Johanniskraut) is considered a noxious weed in many
countries, but its medicinal use dates back to ancient Greece. In modern
medicine, standardized Hypericum extracts are commonly used for treatment of
mild depression and anxiety disorders, and in some countries (e.g. Germany) more
so than synthetic antidepressant medication. A note of caution: the treatments
may interfere with other medications by influencing the metabolism or activity
of other drugs,
and many of these effects might be caused by influences of the hyperforins on
drug-metabolizing cytochrome P450 activities. Hypericin deposited in the skin may lead to extreme light-sensitivity (burns
already after short sunlight exposure). There are a few citations for this text
in another page, dealing with hyperforin
and a proposal for its biosynthesis.
Overviews on Hypericum perforatum
can be found in these Wikipedia pages:
English:
St_John's wort;
Deutsch:
Echtes Johanniskraut
This page focusses on
Hypericin, one of the main components of the extracts.
And here something a bit
unexpected: recent work described that an endophytic fungus isolated from the
stems of Hypericum perforatum contained hypericin and also the precursor
emodin (Kusari
et al., 2008). The same group also described previously that an endophytic
fungus from Nothapodytes foetida produced
camptothecin, an interesting
anticancer drug (Puri et al., 2005).
The authors argued in both cases that such fungal cultures may be nice sources
for production of these important natural products. These findings raise some
intriguing questions on the presence and nature of the biosynthetic genes in
both plants and fungi, and it will be exciting to see what the molecular basis
in the fungi is.
A key enzyme in the
biosynthesis of the backbone should be a polyketide synthase carrying out
seven condensations, producing an octaketide that is processed into emodin
anthrone: see the model for the biosynthesis in Fig. 1 below . It always was an interesting possibility that a type III PKS is responsible,
i.e. a protein from the large superfamily of chalcone synthase related proteins.
Fig. 1. Model for the Biosynthesis of Hypericin in
Hypericum perforatum. The basic scheme is from the book of
Dewick (1997).
Recent work
(Karppinen
and Hohtola, 2008) described excellent
candidates for type III PKS in the
biosynthesis of hyperforin and hypericin in Hypericum perforatum : HpPKS1 and HpPKS2. The pattern of
tissue-specific expression showed that HpPKS1 expression correlated with hyperforin biosynthesis, while HpPKS2 was a candidate for a PKS in hypericin
formation. The two proteins are also in my
general relationship of plant type III PKS: more...,
and a type III PKS was proposed for the key reaction in hyperforin biosynthesis:
more...
Now the same group published
the functional characterization of HpPKS2 after expression of a recombinant
protein in E. coli (Karppinen
et al., 2008). In many ways the results are comparable with those obtained
with another octaketide synthase (OKS), the enzyme cloned from Aloe arborescens
(more...). In both cases the
enzymes were capable of carrying out the predicted seven condensations, but in both cases the expected end products were not obtained; the
prominent results were the derailment products SEK4 and SEK4b. There seems to be
an interesting difference between the two enzymes: the Aloe protein used
preferentially malonyl-CoA, but had also activity with acetyl-CoA, although
apparently not so high. The Hypericum enzyme was apparently only tested with
acetyl-CoA, but indirect evidence seems to argue that malonyl-CoA was not a good
substrate: this was concluded from the failure to find SEK4 and SEK4b in
incubations with other starter substrates. Figure 2 (below)
illustrates the results with the enzyme from Hypericum perforatum.
Fig. 2. Activity of an octaketide synthase (OKS) cloned from Hypericum perforatum with acetyl-CoA
as starter substrate.
The colours indicate the carbon atoms introduced by the seven condensation
reactions. The products in vitro are SEK4 and SEK4b. The reasons for such
derailment products are not obvious: there are no obvious tailoring enzymes
(e.g. reductases) necessary for the formation of the expected product, emodin
anthrone.
With the Aloe enzyme this
failure could be explained by arguing that there should be a reduction step
somewhere in the formation of Chrysophanol anthrone, the expected product (more...),
but it remains unclear what the problem with the Hypericum enzyme might be
because there is no obvious need for a tailoring reaction. However, the
formation of the end product does not just involve seven condensations, but also
a complex subset of other reactions, e.g. aldol condensations, aromatisations,
and decarboxlation of the terminal carboxyl group. The order of these reactions
is pretty unclear, and there may well be functions that do not work properly
in vitro or that even might require unknown helper activities (other
enzymes?).
Like many other
type III PKS (more...), the
H. perforatum enzyme also accepted a variety of other starter CoA-esters,
synthesizing a quite large array of tri- to hepta-ketide products (i.e. two to
six condensations). I myself
found it quite intriguing that in some cases these were not simply pyrones (as
typical for many of these enzymes), but
also phloroglucinols, i.e. products arising from a chalcone synthase type
ring-folding. I did not check that in more detail with other enzymes, but
remember that phloroglucinols could also be formed from unphysiological
substrates by the type III PKS RppA from the bacterium Streptomyces griseus
; the physiological product is a naphtalene backbone (Funa
et al., 2002).
Very good additional arguments for
assigning the function of HpPKS2 to hypericin biosynthesis, however, are provided in this publication.
It has been known for a while that
dark glands in the aerial parts are characteristic for Hypericum perforatum,
and it is believed that they are the places of biosynthesis and deposition of
hypericin in H. perforatum (Zobayed et
al., 2006; Kornfeld et al.,
2007) and related species (Piovan
et al., 2004). The authors now showed by in situ RNA hybridizations that
the HpPKS2 transcripts correlated nicely with the spezialized tissues
accumulating hypericin, thus strengthening their proposal that this type III PKS
is the key enzyme in hypericin biosynthesis.
Links to
the pages: Enzymes with more than three condensations
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References
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Karppinen, K., Hokkanen, J., Mattila, S., Neubauer, P., Hohtola, A.,
2008b. Octaketide-producing type III polyketide synthase from
Hypericum perforatum is expressed in dark glands accumulating
hypericins. FEBS Journal 275, 4329-4342.
Hypericins are biologically active constituents of Hypericum
perforatum (St John's wort). It is likely that emodin anthrone, an
anthraquinone precursor of hypericins, is biosynthesized via the
polyketide pathway by type III polyketide synthase (PKS). A PKS from
H. perforatum, HpPKS2, was investigated for its possible involvement
in the biosynthesis of hypericins. Phylogenetic tree analysis revealed
that HpPKS2 groups with functionally divergent non-chalcone-producing
plant-specific type III PKSs, but it is not particularly closely related
to any of the currently known type III PKSs. A recombinant HpPKS2
expressed in Escherichia coli resulted in an enzyme of ca. 43 kDa.
The purified enzyme catalysed the condensation of acetyl-CoA with two to
seven malonyl-CoA to yield tri- to octaketide products, including
octaketides SEK4 and SEK4b, as well as heptaketide aloesone. Although
HpPKS2 was found to have octaketide synthase activity, production of
emodin anthrone, a supposed octaketide precursor of hypericins, was not
detected. The enzyme also accepted isobutyryl-CoA, benzoyl-CoA and
hexanoyl-CoA as starter substrates producing a variety of tri- to
heptaketide products. In situ RNA hybridization localized the
HpPKS2 transcripts in H. perforatum leaf margins, flower petals
and stamens, specifically in multicellular dark glands accumulating
hypericins. Based on our results, HpPKS2 may have a role in the
biosynthesis of hypericins in H. perforatum but some additional
factors are possibly required for the production of emodin anthrone
in vivo.
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Karppinen, K., Hohtola, A., 2008. Molecular
cloning and tissue-specific expression of two cDNAs encoding polyketide
synthases from Hypericum perforatum. Journal of Plant Physiology
165, 1079-1086.
Two previously
uncharacterized cDNAs encoding for polyketide synthases (PKSs), designated
as HpPKS1 and HpPKS2, were isolated from Hypericum perforatum. The
full-length HpPKS1 was 1573bp containing an open reading frame (ORF) of
1161bp encoding for a 386 amino acid protein. The full-length cDNA of
HpPKS2 was 1559bp with an ORF of 1182bp encoding for a 393 amino acid
protein. The highly conserved catalytic amino acid residues common to
plant-specific PKSs were preserved in both genes. HpPKS1 and HpPKS2
exhibited distinct tissue-specific expression patterns in H. perforatum.
The HpPKS1 expression was highest in flower buds and lowest in root
tissues. The expression of HpPKS2 was found to be high in flower buds and
leaf margins and low in leaf interior parts, stems and roots. The
expression of the HpPKS1 was found to correlate with the concentrations of
hyperforin and adhyperforin while the expression of HpPKS2 showed
correlation with the concentrations of hypericins and pseudohypericins in
H. perforatum tissues.
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Bais, H. P., Vepachedu, R., Lawrence, C. B.,
Stermitz, F. R., Vivanco, J. M., 2003. Molecular and biochemical
characterization of an enzyme responsible for the formation of hypericin in
St. John's Wort (Hypericum perforatum L.). Journal of Biological
Chemistry 278, 32413-32422.
A
major gene termed Hyp-1 encoding for hypericin (HyH) biosynthesis was
cloned and characterized from Hypericum perforatum (St. John's
wort) cell cultures. H. perforatum leaves are widely used as an
herbal remedy in the treatment of mild to moderate depression. Hypericin,
a photosensitive and red-colored naphthodianthrone, has been reported as
the bioactive compound responsible for reversing the depression symptoms.
In this study a novel red-color-based colony screening method for
examining a cDNA library (-TriplEX2) derived from H. perforatum
cell cultures revealed the gene responsible for hypericin biosynthesis
after the administration of emodin, a precursor of hypericin. The
selected clones were expressed in Escherichia coli (BM 25.8 line)
and were further screened for biosynthesis of emodin to hypericin, which
resulted in an 84.6% conversion. The full-length cDNA sequence of Hyp-1
is 782 nucleotides in length with an open reading frame of 477
nucleotides coding for a protein of 159 amino acids, with a 45.1%
homology to Bet.v.1 class allergens. Reverse transcriptase-PCR analysis
showed high levels of Hyp-1 transcripts in dark-grown cell cultures
compared with the levels in light-grown cell cultures and leaves.
Southern blot analysis showed the presence of a single Hyp-1 gene in
H. perforatum. Furthermore, Hyp-1 was expressed with a His6 affinity
tag linked to its N terminal region using the expression vector pET-28a,
and the recombinant Hyp-1 protein was able to convert HyH from emodin
under in vitro conditions. HyH product inhibition was observed with
emodin analogues, rhein, rhein methyl ester, and DNA3-55-1. Our results
demonstrate a direct and complex conversion of emodin to HyH that is
solely catalyzed by Hyp-1, a Bet.v.1 class allergen from H.
perforatum.
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Dewick, P. M., 1997. Medicinal Natural Products - A
Biosynthetic Approach. John Wiley & Sons, Chichester.
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Funa, N., Ohnishi, Y.,
Ebizuka, Y., Horinouchi, S.: Properties and substrate specificity of RppA, a
chalcone synthase-related polyketide synthase in Streptomyces griseus. Journal of Biological Chemistry 277, 4628-4635 (2002).
RppA, a chalcone synthase-related polyketide synthase (type
III polyketide synthase) in the bacterium Streptomyces griseus,
catalyzes the formation of 1,3,6,8-tetrahydroxynaphthalene (T4HN) from five
molecules of malonyl-CoA. The Km value for malonyl-CoA and
the kcat value for T4HN synthesis were determined to be 0.93
+/- 0.1 µM and 0.77 +/- 0.04 min-1, respectively. RppA accepted
aliphatic acyl-CoAs with the carbon lengths from C4 to C8 as starter
substrates and catalyzed sequential condensation of malonyl-CoA to yield
alpha-pyrones and phloroglucinols. In addition, RppA yielded a hexaketide,
4-hydroxy-6-(2',4',6'-trioxotridecyl)-2-pyrone, from octanoyl-CoA and five
molecules of malonyl-CoA, suggesting that the size of the active site cavity
of RppA is larger than any other chalcone synthase-related enzymes found so
far in plants and bacteria. RppA was also found to synthesize a C-methylated
pyrone, 3,6-dimethyl-4-hydroxy-2-pyrone, by using acetoacetyl-CoA as the
starter and methylmalonyl-CoA as an extender. Thus, the broad substrate
specificity of RppA yields a wide variety of products.
more...
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Kornfeld, A., Kaufman, P. B., Lu,
C. R., Gibson, D. M., Bolling, S. F., Warber, S. L., Chang, S. C.,
Kirakosyan, A., 2007.
The
production of hypericins in two selected Hypericum perforatum shoot
cultures is related to differences in black gland structure. Plant
Physiology and Biochemistry 45, 24-32.
In vitro
shoot cultures of Hypericum perforatum derived from wild
populations grown in Armenia have a wide variation of hypericin and
pseudohypericin metabolite content. We found that a germ line denoted as
HP3 produces six times more hypericin and fourteen times more
pseudohypericin than a second line labeled HP1. We undertook a
structural comparison of the two lines (HP1 and HP3) in order to see if
there are any anatomical or morphological differences that could explain
the differences in production of these economically important
metabolites. Analysis by LM (light microscopy), SEM (scanning electron
microscopy), and TEM (transmission electron microscopy) reveals that the
hypericin/pseudohypericin-containing black glands located along the
margins of the leaves consist of a peripheral sheath of flattened cells
surrounding a core of interior cells that are typically dead at maturity.
The peripheral cells of the HP3 glands appear less flattened than those
of the HP1 glands. This may indicate that the peripheral cells are
involved in hypericin/pseudohypericin production. Furthermore, we find
that these peripheral cells undergo a developmental transition into the
gland's interior cells. The fact that the size of the peripheral cells
may correlate with metabolite production adds a new hypothesis for the
actual site of hypericin synthesis.
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Kusari, S., Lamshöft, M., Zühlke, S., Spiteller,
M., 2008. An endophytic fungus from Hypericum perforatum that
produces hypericin. Journal of Natural Products 71, 159-162.
For the first time, an endophytic fungus has been isolated from the
stems of the medicinal herb Hypericum perforatum (St. John's
Wort). The fungus produced the napthodianthrone derivative hypericin (1)
in rich mycological medium (potato dextrose broth) under shake flask and
bench scale fermentation conditions. Emodin (2) was also produced
simultaneously by the fungus under the same culture conditions. We
propose 2 as the main precursor in the microbial metabolic pathway to 1.
The fungus was identified by morphology and authenticated by 28S (LSU)
rDNA sequencing. Compounds 1 and 2 were identified by LC-HRMS, LC-MS/MS,
and LC-HRMS/MS and confirmed by comparison with authentic standards. In
bioassays with a panel of laboratory standard pathogenic control strains,
including fungi and bacteria, both fungal 1 and 2 possessed
antimicrobial activity comparable to authentic standards. This
endophytic fungus has significant scientific and industrial potential to
meet the pharmaceutical demands for 1 in a cost-effective, easily
accessible, and reproducible way.
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Piovan, A., Filippini, R., Caniato, R., Borsarini,
A., Maleci, L. B., Cappelletti, E. M., 2004. Detection of hypericins in the
"red glands" of Hypericum elodes by ESI-MS/MS. Phytochemistry 65,
411-414.
The
biologically active naphthodianthrones hypericin and pseudohypericin
were detected by electrospray ionization mass spectrometry (ESI-MS/MS)
in microsamples from the sepals of Hypericum elodes (Hypericaceae)
containing the so-called "red glands", i.e. stipitate glands with
red-coloured heads. The occurrence of hypericins in the red glands of
H. elodes supports the taxonomic position of the section Elodes
within the genus Hypericum and provides evidence that the ability of
carrying out the biosynthetic pathway leading to the naphthodianthrone
compounds, rather than the absolute amounts produced, should be regarded
as a chemical marker of the phylogenetically more advanced sections of
genus Hypericum. The biologically active phloroglucinol derivatives
hyperforin and adhyperforin, so far found only in H. perforatum,
were also detected and evidence for their localization in the sepal
secretory canals with large lumen, is given.
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Puri, S. C., Verma, V., Amna, T., Qazi, G. N.,
Spiteller, M., 2005. An endophytic fungus from Nothapodytes foetida
that produces Camptothecin. Journal of Natural Products 68, 1717-1719.
A fungal
endophytic isolate, camptothecin, has been isolated from the inner bark
of the plant Nothapodytes foetida from the Western coast of India.
The fungus, which belongs to the family Phycomycetes, produced the
anticancer drug lead compound camptothecin (1) when grown in a synthetic
liquid medium (Sabouraud broth) under shake flask and bench scale
fermentation conditions. Compound 1 was identified by means of
chromatographic and spectroscopic methods. It was also compared with an
authentic example for its biological activity against a number of human
cancer cell lines. Isolation of an organism producing 1 and its
fermentation may, in the future, provide an easily accessible source for
the production of this anticancer drug precursor molecule.
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Zobayed, S. M. A., Afreen, F., Goto, E., Kozai,
T., 2006. Plant-environment interactions: accumulation of hypericin in dark
glands of Hypericum perforatum. Annals of Botany 98, 793-804.
A
significant presence of dark glands accompanying the highest
concentrations of Hy-G was observed in the stamen tissues more than in
any other organ of H. perforatum. A linear relationship between
the number of dark glands and net photosynthetic rate of the leaf and
Hy-G concentration in the leaf tissue was also established. A very high
concentration of Hy-G was measured in the dark-gland tissues, but in the
tissues without any dark glands it was almost absent. The presence of
emodin, a precursor of Hy-G, at a high concentration in the dark-gland
tissues, and its absence in the surrounding tissues was also observed,
suggesting that the site of biosynthesis of Hy-G is in the dark-gland
cells. A significantly low concentration of Hy-G (occasionally
non-detectable) was measured in the xylem sap of the stem tissues. The
dark-gland tissues collected from leaves, stems or flowers contained
similar concentrations of Hy-G. The concentration of Hy-G in various
organs of H. perforatum plants is dependent on the number of dark
glands, their size or area, not on the location of the dark glands on
the plant. The study provides the first experimental evidence that Hy-G
is synthesized and accumulates in dark glands.
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