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(Last
modification: 09. March 2009)
Fünf
Kondensationsreaktionen:
Hexaketid-Synthase (HKS) in der Plumbagin Biosynthese
(Springob
et al., 2007,
Jindaprasert
et al., 2008).
Etliche Pflanzen enthalten
Naphthochinone wie z.B. Plumbagin (Thomson,
1957), und
Plumbago indica
ist ein
wohluntersuchtes Beispiel, weil die Pflanze vielfältige medizinische
Anwendungen in Südost-Asien hat. Vor langer Zeit wurde bereits gezeigt, dass
Plumbagin aus Acetyl-Einheiten gebildet wird (Durand
and Zenk,
1974).
Daraus konnte man vermuten, dass Acetyl-CoA als Starter dient, und dass fünf
Kondensationsreaktionen mit Malonyl-CoA durchgeführt werden. Nach der Entdeckung
dass pflanzliche Typ III PKS mehr als drei Kondensationen durchführen können waren solche Enzyme Kandidaten für eine solche Reaktion.
Dies wurde kürzlich mit Typ III PKS aus
Plumbago indica (Springob
et al., 2007) und Drosophyllum lusitanicum untersucht (Jindaprasert
et al., 2008). Die Proteine waren nur 47-60% identisch mit anderen Typ
III Enzymen, und dieser niedrige Wert war schon ein gutes Indiz, dass es sich
hier nicht um noch eine CHS, sondern um andere Enzym handelte.
Detaillierte Untersuchungen mit rekombinanten Proteinen
(Expression in E. coli) zeigten dann, dass die Enzyme mit Acetyl-CoA aktiv
waren, wie vorhergesagt. Aber Plumbagin war nicht unter den Produkten; es waren
stattdessen Pyrone, die man nach zwei, drei, vier, oder sogar fünf
Kondensationsreaktionen erwarten würde. Das Schema unten zeigt die Reaktion, die
man in vivo erwarten würde, und die Produkte, die man in vitro
tatsächlich erhielt. Wenn man sich die postulierte Biosynthese ansieht wird auch
offensichtlich, was höchstwahrscheinlich das Problem in vitro ist: Die
Reaktionen zu Plumbagin müssen irgendwo einen Reduktionsschritt enthalten
(Symbol (H) in roter Box in der Schemazeichnung), und
der konnte natürlich nicht von den rekombinanten PKS durchgeführt werden. Diese
Probleme mit Fehlen einer
postulierten
Reduktase sind natürlich wohlbekannt, da
in den letzten Jahre mehrere andere Beispiele dafür beschrieben wurden. Dazu gehören z.B.
die
Oktaketid-Synthase aus
Aloe arborescens ebenso wie
die Stilbencarboxylat-Synthasen
aus Hydrangea
macrophylla (Eckermann et
al., 2003) und dem Lebermoos
Marchantia polymorpha (Schröder et al.,
unpubliziert, aber in unserer Website diskutiert:
Mehr...). Das Fehlen eines (oder mehrerer) solcher oder weiterführender Enzyme
ist vermutlich auch verantwortlich für die Probleme mit der in vitro
Synthese eines Produktes, welches für eine Typ III PKS
aus
Wachendorfia thyrsiflora vorhergesagt wurde (Brand
et al., 2006).
Wie von anderen Typ III PKS bekannt, akzeptierte die Enzyme
auch eine grosse
Anzahl anderer Substrate, einschliesslich solch grosser Moleküle wie
4-Coumaroyl-CoA und Cinnamoyl-CoA, die Standardsubstrate von Chalcon- und
Stilbensynthasen. Jedoch, die Produkte waren immer nur die Tri- und Tetraketid-Pyrone nach zwei und
drei Kondensationsreaktionen (mit 4-Coumaroyl-CoA als Substrat:
Bisnoryangonin
und
4-Coumaroyltriacetat).
Die verfügbaren Daten lassen vermuten, dass diese Enzyme tatsächlich an der Plumbagin-Biosynthese beteiligt
sind, aber der
endgültige Nachweis muss wohl anders geführt werden (transgene Expression?
Gen-Deletion?).
Zum
Seitenanfang
Links zu den
einzelnen Seiten: Enzyme mit mehr als drei Kondensationen
Zum
Seitenanfang
.
Zitate
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Jindaprasert, A., Springob, K., Schmidt, J., De-Eknamkul, W., Kutchan, T.
M., 2008.
Pyrone
polyketides synthesized by a type III polyketide synthase from Drosophyllum
lusitanicum. Phytochemistry 69, 3043-3053.
To isolate
cDNAs involved in the biosynthesis of acetate-derived naphthoquinones in
Drosophyllum lusitanicum, an expressed sequence tag analysis was
performed. RNA from callus cultures was used to create a cDNA library from
which 2004 expressed sequence tags were generated. One cDNA with similarity to
known type III polyketide synthases was isolated as full-length sequence and
termed DluHKS. The translated polypeptide sequence of DluHKS showed 51-67%
identity with other plant type III PKSs. Recombinant DluHKS expressed in
Escherichia coli accepted acetyl-coenzyme A (CoA) as starter and carried
out sequential decarboxylative condensations with malonyl-CoA yielding
alpha-pyrones from three to six acetate units. However, naphthalenes, the
expected products, were not isolated. Since the main compound produced by
DluHKS is a hexaketide alpha-pyrone, and the naphthoquinones in D.
lusitanicum are composed of six acetate units, we propose that the enzyme
provides the backbone of these secondary metabolites. An involvement of
accessory proteins in this biosynthetic pathway is discussed.
Zurück
-
Springob, K., Samappito, S.,
Jindaprasert, A., Schmidt, J., Page, J. E., De-Eknamkul, W., Kutchan, T. M.,
2007. A polyketide synthase of Plumbago indica that catalyzes the
formation of hexaketide pyrones. FEBS Journal 274, 406-417.
Plumbago
indica L. contains naphthoquinones that are derived from six acetate
units. To characterize the enzyme catalyzing the first step in the
biosynthesis of these metabolites, a cDNA encoding a type III polyketide
synthase (PKS) was isolated from roots of P. indica. The translated
polypeptide shared 47-60% identical residues with PKSs from other plant
species. Recombinant P. indica PKS expressed in Escherichia coli
accepted acetyl-CoA as starter and carried out five decarboxylative
condensations with malonyl coenzyme A (-CoA). The resulting hexaketide was not
folded into a naphthalene derivative. Instead, an alpha-pyrone,
6-(2',4'-dihydroxy-6'-methylphenyl)-4-hydroxy-2-pyrone, was produced. In
addition, formation of alpha-pyrones with linear keto side chains derived from
three to six acetate units was observed. As phenylpyrones could not be
detected in P. indica roots, we propose that the novel PKS is involved
in the biosynthesis of naphthoquinones, and additional cofactors are probably
required for the biosynthesis of these secondary metabolites in vivo.
Zurück zum Text
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Brand, S.,
Hölscher, D., Schierhorn, A., Svatos, A., Schröder, J., Schneider, B., 2006.
A type III polyketide synthase from Wachendorfia thyrsiflora
and its role in diarylheptanoid and phenylphenalenone biosynthesis. Planta
224, 413-428.
Chalcone synthase (CHS) related type III plant polyketide synthases (PKSs) are likely to be involved in the biosynthesis of diarylheptanoids (e.g. curcumin and polycyclic phenylphenalenones), but no such activity has been reported. Root cultures from Wachendorfia thyrsiflora (Haemodoraceae) are a suitable source to search for such enzymes because they synthesize large amounts of phenylphenalenones, but no other products that are known to require CHSs or related enzymes (e.g. flavonoids or stilbenes). A homology-based RT-PCR strategy led to the identification of cDNAs for a type III PKS sharing only approximately 60% identity with typical CHSs. It was named WtPKS1 (W. thyrsiflora polyketide synthase 1). The purified recombinant protein accepted a large variety of aromatic and aliphatic starter CoA esters, including phenylpropionyl- and side-chain unsaturated phenylpropanoid-CoAs. The simplest model for the initial reaction in diarylheptanoid biosynthesis predicts a phenylpropanoid-CoA as starter and a single condensation reaction to a diketide. Benzalacetones, the expected release products, were observed only with unsaturated phenylpropanoid-CoAs, and the best results were obtained with 4-coumaroyl-CoA (80% of the products). With all other substrates, WtPKS1 performed two condensation reactions and released pyrones. We propose that WtPKS1 catalyses the first step in diarylheptanoid biosynthesis and that the observed pyrones are derailment products in the absence of downstream processing proteins.Sonderdruckanfrage
Zur
Besprechung dieses Enzyms (andere Seite), oder
zurück zum Text
-
Durand, R., Zenk, M. H., 1974. The
homogentisate ring-cleavage pathway in the biosynthesis of acetate-derived
naphthoquinones of the Droseraceae.
Phytochemistry 13, 1483-1492.
Photosynthesis experiments with 14CO2 established that of 16
Droseraceae species tested Drosophylum lusitanicum incorporated the
highest amount of label into plumbagin
(2-methyl-5-hydroxy-1,4-naphthoquinone). Tyrosine-[ß-14C] fed to
Drosophyllum was shown to label plumbagin efficiently (20%
incorporation). Extensive chemical degradation of the labeled naphthoquinone
showed, however, that the incorporation of tyrosine was indirect, the label
being distributed throughout the molecule. It was established that plumbagin
and the closely related 7-methyljuglone are biosynthesized via the
acetate-polymalonate pathway. Tyrosine is broken down to acetate in this
tissue via the homogentisate pathway, which was demonstrated by feeding and
incorporation of label into plumbagin of intermediates such as
homogentisate-[14C], maleyl- and fumarylacetoacetate-[14C].
Simultaneous application of tyrosine-[ß-14C] and a,a'-bipyridyl,
an inhibitor of the homogentisate oxigenase, led to an accumulation of
homogentisate-[14C] within the tissue. The degradation of
tyrosine to acetate by Drosophyllum is not due to epiphytic bacteria
since ring cleavage of tyrosine and formation of plumbagin from breakdown
products occurred both within sterile grown plants and sterile cell
suspension cultures. In tissue kept in darkness, plumbagin undergoes a slow
turnover with a half life of about 400 hr.
Zurück zum Text
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Eckermann,
C., Schröder, G., Eckermann, S., Strack, D., Schmidt, J., Schneider, B.,
Schröder, J., 2003.
Stilbenecarboxylate biosynthesis: a new function in the family of chalcone
synthase related proteins. Phytochemistry 62, 271-286.
Chalcone (CHS), stilbene (STS) synthases, and related proteins are key enzymes in the biosynthesis of many secondary plant products. Precursor feeding studies and mechanistic rationalization suggest that stilbenecarboxylates might also be synthesized by plant type III polyketide synthases; however, the enzyme activity leading to retention of the carboxyl moiety in a stilbene backbone has not yet been demonstrated. Hydrangea macrophylla L. (Garden Hortensia) contains stilbenecarboxylates (hydrangeic acid and lunularic acid) that are derived from 4-coumaroyl and dihydro-4-coumaroyl starter residues, respectively. We used homology-based techniques to clone CHS-related sequences, and the enzyme functions were investigated with recombinant proteins. Sequences for two proteins were obtained. One was identified as CHS. The other shared 65-70% identity with CHSs and other family members. The purified recombinant protein had stilbenecarboxylate synthase (STCS) activity with dihydro-4-coumaroyl-CoA, but not with 4-coumaroyl-CoA or other substrates. We propose that the enzyme is involved in the biosynthesis of lunularic acid. It is the first example of a STS-type reaction that does not lose the terminal carboxyl group during the ring folding to the end product. Comparisons with CHS, STS, and a pyrone synthase showed that it is the only enzyme exerting a tight control over decarboxylation reactions. The protein contains unusual residues in positions highly conserved in other CHS-related proteins, and mutagenesis studies suggest that they are important for the structure or/and the catalytic activity. The formation of the natural products in vivo requires a reducing step, and we discuss the possibility that the absence of a reductase in the in vitro reactions may be responsible for the failure to obtain stilbenecarboxylates from substrates like 4-coumaroyl-CoA.
Sonderdruckanfrage
Zurück zum Text, oder zur
Diskussion dieser Enzyme (andere Seite)
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Thomson, R.H., 1957. Naturally
Occuring Quinones. Butterworth Scientific, London.
Zurück zum Text
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