|
(Last
modification: 10. July 2010)
Typ III PKS:
Nebenprodukte, Promiskuität, Funktionelle Identifizierung
Praktisch alle Typ III Polyketidsynthasen (PKS) haben in vitro ein
wenig irritierende Eigenschaften, d.h. als rekombinante Proteine nach Expression in E.
coli oder anderen Wirten (Hefe, tierische Zellen, Insektenzellen):
Deswegen:
-
3. Die funktionelle
Identifizierung neuer Mitglieder der Familie ist nicht immer einfach:
Mehr....
-
4. Und hier gibt es auch einige Beispiele: Unbekannte oder unklare physiologische
Rolle, 'Orphan
PKS': Mehr...
Diese Themen sollen hier kurz besprochen werden. Die Diskussion wird sich
beispielhaft meist
auf Chalcon- (CHS), Stilben- (STS), und Stilbencarboxylat- (STCS) Synthasen
konzentrieren, aber die Problematik ist bei praktisch allen Typ III Proteinen
gleich.
1.
Nebenprodukte
In der Regel synthetisieren Chalconsynthasen (CHS), Stilbensynthasen (STS), und
Stilbencarboxylatsynthasen (STCS) in vitro nicht nur die erwarteten Produkte,
sondern auch Nebenprodukte, die als Derivate von Reaktions-Intermediaten erklärt
werden können. Die Abbildung fasst in den Boxen die Nebenprodukte zusammen, die
üblicherweise erhalten werden, und zwar mit 4-Coumaroyl-CoA als repräsentativem Substrat. Dies sind
typischerweise
Benzalaceton (eine Kondensation), Arylpyrone (eine
Kondensation), Styrylpyrone (z.B. Bisnoryangonin, zwei Kondensationen), und
4-Coumaroyltriacetsäure-Lacton (CTAL, drei
Kondensationen, auch ein Pyron).
Reactions of CHS (chalcone synthase), STS (stilbene
synthase), and STCS (stilbenecarboxylate synthase), and byproducts in vitro.
The reactions are given for the prototype substrate 4-coumaroyl-CoA. The colours
mark the three condensation reactions. The byproducts are boxed. Most abundant
are the pyrone derailment products after two and three condensations (Bisnoryangonin
and 4-Coumaroyltriacetic acid, CTAL).
Benzalaceton, Arylpyrone, und Bisnoryangonin
wurden als Nebenprodukte der CHS schon bei der Entdeckung und ersten
Charakterisierung der CHS beschrieben (Kreuzaler and Hahlbrock,
1975a, 1975b;
Hrazdina et al., 1976;
Saleh et al., 1978).
Interessanterweise sind zumindest Benzalaceton und Bisnoryangonin auch Naturstoffe in Pflanzen: Benzalaceton ist die
Vorstufe zur Hauptaromakomponente in der
Himbeere (Borejsza-Wysocki
and Hrazdina, 1994;
1996);
es wird durch eine Typ III PKS synthetisiert (Hrazdina and Zheng,
2006; Zheng and Hrazdina,
2008, mehr...), und in einem
Rhabarber wird Benzalaceton auch durch eine Typ III PKS gebildet (Rheum palmatum, Abe et al.,
2001; 2003,
mehr...). Bisnoryangonin gibt
es in vielen Pflanzen (Beckert
et al., 1997; Übersicht in:
Schröder, 1999), aber hier steht der Nachweis wohl noch aus, dass es durch
eine Typ III PKS synthetisiert wird (mehr...).
4-Coumaroyltriacetsäure-Lacton (CTAL) dagegen wurde relativ spät entdeckt (Yamaguchi
et al., 1999), und es wurde als das physiologische Produkt einer Typ III PKS aus
Hydrangea macrophylla var. thunbergii diskutiert (Akiyama
et al., 1999).
Die Nebenprodukte kann man in praktisch allen CHS,
STS, und STCS Reaktionen finden, wenn man genau hinschaut: Die Pyrone
Bisnoryangonin und besonders das CTAL gehen bei der Produktaufarbeitung leicht
verloren. Besonders
das Letztere ist kaum zu finden, wenn, wie in vielen Fällen üblich, die Produkte
der Enzymreaktionen bei pH 7 (oder höher) mit Ethylacetat extrahiert werden.
Noch schlimmer mit Abbruch-Reaktionen wird es, wenn man unphysiologische
Substrate verwendet, s. nächstes Kapitel: Typischerweise können alle Typ III PKS viele
Substrate akzeptieren, und zwar oft genauso gut wie die physiologischen
Substrate. Aber meist kommen dabei nicht die erwarteten Produkte heraus, da die
Reaktionen frühzeitig abgebrochen werden. Beispiele: Bei CHS wird die Reaktion
mit unphysiologischen Substraten meist nach zwei Kondensationen abgebrochen (->
Pyron-Produkt, s. Bisnoryangonin-Typ), oder wenn schon drei Kondensationen
möglich sind, gelingt die Faltung zum Chalcon-Typ Produkt nicht mehr (->
Pyron-Produkt, s. CTAL-Typ). Zur Beachtung: Genau die gleichen
Nebenprodukte erhält man mit STS oder STCS, s. Abbildung oben! Wenn man also
solche Produkte mit einem neu charakterisierten Protein erhält: Sind das nun die
'wirklichen' Produkte oder nur Nebenprodukte von entweder CHS, STS, oder STCS?
Und war das verwendete Substrat überhaupt das physiologische? Die Antwort ist
nicht immer leicht.
Und auch dies noch: Sogar die Spezifität
der Ringfaltung zum Chalcon und Stilben ist nicht absolut: Es gibt Berichte,
dass ein paar Prozente der CHS-Produkte tatsächlich Stilbene waren, und dass
umgekehrt ein paar Prozente der STS-Produkte Chalcone waren (Yamaguchi et al.,
1999; Suh et al., 2000).
Zum Seitenanfang
2.
Substrat-Promiskuität
Alle diese Enzyme sind nicht sehr substratspezifisch. CHS, STS und STCS zum Beispiel
akzeptieren alle CoA-Ester die so etwa die gleiche Grösse wie das physiologische
Substrat 4-Coumaroyl-CoA haben, und die Aktivitäten mit ihnen können genau so
hoch sein. Dies wurde bereits sehr früh beschrieben, und eines der ersten
schönen Beispiele ist die Arbeit von
Schüz et al. (1983); sie zeigte bereits damals, dass Benzoyl-CoA,
Hexanoyl-CoA, und andere aliphatische CoA-Ester excellente Substrate der CHS
sind. Diese Promiskuität wurde durch alle späteren Untersuchungen mit anderen
CHS bestätigt (wenn es denn überhaupt untersucht wurde). Auch bestätigt wurde,
dass die Reaktionen in den meisten Fällen vorzeitig abgebrochen werden, oder
dass die Faltung zum Chalcon-Typ Produkt nicht erreicht wird (wie bereits oben
besprochen).
Manchmal führt dies zu erstaunlichen Ergebnissen.
Zum Beispiel: Das physiologische Substrat der
Pentaketid-Synthase (PCS) und
der
Oktaketid-Synthase (OKS) aus
Aloe arborescens ist vermutlich Malonyl-CoA (oder vielleicht
Acetyl-CoA), und die Enzyme führen damit vier (PCS) oder sieben (OKS)
Kondensationen durch. Aber in vitro akzeptieren sie auch sehr gut viel
grössere Substrate, wie zum Beispiel CoA-Ester von langkettigen Fettsäuren, und
sogar auch 4-Coumaroyl-CoA, das Prototyp-Substrat der
CHS und STS. Die Produkte sind die Pyrone von zwei oder drei
Kondensationsreaktionen. Mit 4-Coumaroyl-CoA sind das Bisnoryangonin und CTAL;
Chalcone oder Stilbene wurden nicht gefunden. Eigentlich sollte man in
solchen Fällen früher oder später zusätzliche Evidenz für die vorgeschlagene
physiologische Funktion der Enzyme beibringen (s. unten).
Und es kann noch komplizierter werden. Die
Typ III PKS sind nicht nur sehr wenig wählerisch mit dem Starter-Substrat, sondern in
vielen Fällen auch nicht mit dem Kettenverlängerer. Standard für diese Enzyme ist Malonyl-CoA,
aber Methylmalonyl-CoA geht in vielen Fällen auch. Als physiologischer Kettenverlängerer wurde es
in einem Falle bereits vorgeschlagen (Schröder
et al., 1998), in der
Biosynthese C-methylierter Flavonoide:
Mehr.... Spätere Experimente lassen
vermuten, dass auch andere Typ III PKS diese Fähigkeit besitzen, zumindest
in vitro (Abe
et al., 2002; Abe et al.,
2003; Abe et al., 2006).
Diese Promiskuität kann auch ganz
interessante Anwendungen haben: Man kann ganz neue Produkte erhalten, wenn man
total artifizielle Substrate anbietet (Morita
et al., 2000; Morita
et al., 2001).
Zum Seitenanfang
3.
Konsequenzen für die
funktionelle Identifizierung neuer Typ
III Enzyme
Die beiden oben besprochenen Eigenschaften haben einige Konsequenzen, wenn Sie
an der Identifizierung der physiologischen Funktion neuer Familien-Mitglieder
interessiert sind. Neue Typ III Enzyme bekommt man meist durch Homologie-basierte
Klonierungs-Strategien (z.B. durch Hybridisierung, RT-PCR mit degenerierten
Primern, oder neuerdings durch 'Genome mining', also durch Homologiesuchen in
Genbanken). Damit kann man einiges finden, welches laut Sequenz wohl in die
Familie der Typ III PKS gehört. Der nächste und kritische Schritt ist dann der Versuch, eine
Funktion zu finden, und wenn möglich die, welche das Protein in der
Pflanze hat.
Zunächst ist in den meisten Fällen der
erste Schritt: Heterologe Expression in einem anderen Wirt (E. coli,
Hefe, Insektenzellen, usw.), Reinigung des Proteins, und Teste mit verschiedenen
Substraten. Die erste Wahl wird hier sicherlich dasjenige sein, welches zu einem
Produkt führt, welches in der untersuchten Pflanze vorhanden ist. Dies muss
nicht gleich der Naturstoff selbst sein; häufig lassen sich in komplizierten
Naturstoffen denkbare PKS-synthetisierte Vorstufen ableiten. Eine halbwegs
sorgfältige Untersuchung wird jedoch auch andere Substrate testen. Ganz sicher sollten
die Prototyp-Substrate der CHS dabei sein, denn Gene (meistens sogar viele:
Genfamilie!) für dieses Enzym sind allgegenwärtig in Pflanzen, und die
Wahrscheinlichkeit ist hoch, dass alle Strategien auch diese Gene finden.
Mit etwas Glück führen diese Experimente zu dem erwünschten Ergebnis: Das neue
Typ III Protein ist nicht einfach eine zusätzliche CHS, sondern macht mit einem anderen Substrat ein
Produkt, welches einem Naturstoff (oder einem Precursor) in der Pflanze zugeordnet werden kann.
Oder es katalysiert mit einem wohlbekannten Substrat eine neuartige Reaktion zu
einem neuen Produkt, und so etwas gibt es in der Pflanze. Dann sind Sie gut dran, sogar wenn auch andere Substrate
akzeptiert werden: Das demonstriert dann wieder einmal die Promiskuität dieser
Enzyme.
Leider ist dies aber häufig nicht so einfach, und es
gibt mehrere Gründe dafür, dass man partout nicht das erwartete Produkt in
vitro erhält. Ein trivialer ist manchmal, dass man das rekombinante Protein
einfach nicht in löslicher Form bekommen kann. Es gibt viele Möglichkeiten, dies
zu optimieren, aber manchmal scheitert man doch daran: Ein vernünftiger Test der
Aktivität ist dann gar nicht erst möglich. Oder das Enzym nimmt zwar das
erwartete Substrat ganz gut, aber schafft es aus irgendwelchen Gründen nicht, die
erwartete Reaktion
ganz durchzuführen (s. die Nebenprodukte oben), und/oder gleichzeitig nimmt es auch
andere Substrate, und die Substrat-Promiskuität verhindert eine klare
Identifizierung des vermutlich physiologischen Substrats. Manchmal bekommt man
perfekt die erwartete Reaktion, aber sie ist der der CHS so ähnlich, dass die
Substrat-Promiskuität es verhindert, die physiologische Rolle festzulegen. Ein
Beispiel dafür ist die
Valerophonsynthase (VPS) aus dem Hopfen:
Mehr.... Es ist wohl immer noch nicht so
ganz klar, welches der vielen klonierten Typ III PKS nun die 'wirkliche' CHS oder VPS
ist: Mehrere der Proteine scheinen beide Aktivitäten zu haben, wenigstens in
vitro. Im übrigen gibt es noch andere Enzyme, die eine
CHS-Typ Ringfaltung mit anderen
Substraten durchführen (mehr...), und das gleiche gilt für die
STS-Typ Ringfaltung (mehr...).
Einer der häufigsten nicht so trivialen Gründe scheint
zu sein, dass die isolierte Typ III PKS allein nicht in der Lage ist, die ganze
Reaktion zu einem erwarteten Endprodukt durchzuführen, da etwas fehlt. Sehr wahrscheinlich ist
dies so in einigen Fällen, die auf anderen Seiten dieser Website besprochen
werden: Entweder fehlt eine postulierte Reduktase (mehr....)
oder es fehlt eine weiterführende Reaktion mit einem weiteren Enzym (mehr.....).
Dann wird es schwierig, und es wird notwendig, andere
unterstützende Evidenz zu bekommen (etwas, das auf längere Sicht sowieso gemacht
werden sollte). Beispiele für Punkte, die helfen können (kein Anspruch auf
Vollständigkeit!):
-
Sehen
Sie sich die Proteinsequenz genauer an:
-
wenn sie 90% oder mehr identisch
mit typischen CHS ist: Dann ist es sehr wahrscheinlich auch eine. Typ III PKS mit
70% oder weniger Identität zu CHS haben in den meisten Fällen auch eine andere
Funktion.
-
Sehen Sie sich die Motive/Aminosäuren an, die bei anderen Typ III PKS
bekannterweise wichtig sind für Substrat- und Produkt-Grösse, und versuchen
Sie eine Interpretation Ihres neuen Proteins. Oder versuchen Sie, Ihr Protein
anhand bekannter 3D-Strukturen zu modellieren (leicht möglich über
Internet-Server, z.B.
Swissmodel). Allerdings, bisher war dies
immer ein 'Erkennen im Nachhinein', d.h. die physiologische Funktion war
bereits bekannt, und man konnte Unterschiede zu anderen Funktionen intelligent
interpretieren.
-
Vorhersagen für neue Aktivitäten aus Sequenz/Motif-Analysen? Sieht nicht so gut aus. In
den meisten Fällen waren solche Sequenz-Analysen nur sehr beschränkte Hilfen
bei der Identifizierung des physiologischen Substrates. Ein typisches
Beispiel: Sogar heutzutage ist es kaum möglich, CHS, STS und STCS durch solche
Sequenz-Analysen oder anhand von Modellen zu unterscheiden. Und es ist sogar
möglich, dass nicht einmal fertige 3D-Strukturen dazu ausreichen (mehr....).
-
Ich möchte hierauf ein bisschen mehr eingehen, aufgrund von Gedanken, die
kürzlich in einem Übersichtsartikel diskutiert wurden (Jiang
et al., 2008): Gibt es Möglichkeiten, die Funktion aus
phylogenetischen/vergleichenden Analysen vorherzusagen?
-
Solche Vorhersagen werden vermutlich nicht
möglich sein für Enzyme aus Moosen, Farnen, und Gymnospermen, weil CHS und
nicht-CHS so dicht in einem Cluster sitzen, dass eine Unterscheidung nicht
möglich ist.
-
Mit Angiospermen ist es ein bisschen
kompliziert. Ein Protein in einem gemeinsamen Cluster mit CHS ist nicht
notwendigerweise eine CHS; es kann alles mögliche andere sein. Beispiele
sind die Fabales: CHS und STS sind in der gleichen Gruppe, und alles Starren
auf Sequenzen oder Motive wird den Unterschied zwischen CHS und STS kaum
erkennen lassen. Andererseits, wenn ein Angiospermen Protein weit weg von
typischen CHS gruppiert, zusammen mit Nicht-CHS, dann sind die Chancen gut,
dass es tatsächlich nicht eine CHS ist. Aber darüber hinaus sind keine
Aussagen über die Funktion möglich: Die Gruppe ist zu heterogen inbezug auf
die Funktion.
-
Mit
Monocotylen ist es auch ein wenig
kompliziert. Die Proteine tendieren dazu, in einer grossen Gruppe abgetrennt
von anderen zu sein. Aber diese Gruppe enthält CHS und Nicht-CHS! Zum
Beispiel ist CHS8 aus Sorghum bicolor tatsächlich eine STS (Yu
et al., 2005), aber ist in der grossen CHS-Gruppe. Andererseits
sind Proteine, die ganz woanders in einer Gruppe sitzen, vermutlich keine
CHS, geradeso wie bei den Angiospermen. Allerdings bezieht sich diese
Aussage bisher nur auf ein Enzym, die Curcuminoidsynthase (CUS), die auf
einer anderen Seite besprochen wird (mehr...).
Die Zukunft wird zeigen, ob solche Verallgemeinerung für Monocotylen Typ
III PKS Bestand hat.
Neu im März 2010: Die Alkylresorcinol-Synthasen (ARS) aus der
Hirse (Sorghum bicolor) und Reis (Oryza sativa) sind
tatsächlich in einer Gruppe, die phylogenetisch weit entfernt ist von den
CHS / STS aus Monokotylen:
mehr...
-
Können
Sie die Enzymaktivität in
vitro in Pflanzenextrakten messen, und ist sie induzierbar?
Dann kann man
zeigen, dass Änderungen der Enzymaktivität mit Änderungen der mRNA
korrelieren; das kann eine gute Argumentationshilfe sein.
-
Ist
Ihre Pflanze genetischen Techniken zugänglich, z.B. Gen-Transfer, incl. RNAi
Strategien?
Versuchen
Sie, die Genfunktion auszuschalten, entweder durch direkten 'knockout' der
Sequenz, oder durch RNAi, oder finden Sie eine Mutante, oder
versuchen Sie, das Gen zur Überexpression durch Einführung zusätzlicher Gene
unter neuer Steuerung bringen. Wenn Ihre Annahme über die Funktion korrekt
ist, sollten sich solche Manipulationen mit Änderungen der Enzymaktivität und
Endprodukte korrelieren lassen. Leider ist aber eine Tatsache, dass viele
Pflanzen mit den interessantesten Naturstoffen mit solchen Techniken nicht
bearbeitet werden können.
-
Ein
Ausweg könnte sein: Exprimieren Sie das isolierte Gen (oder cDNA) in einer
Pflanze, mit der genetische Manipulationen kein Problem sind, z.B. Arabidopsis thaliana, Tabak, usw.
Wenn Sie Glück haben, finden Sie dann
neue Produkte, die den Erwartungen entsprechen. Wenn Sie Pech haben, finden
Sie überhaupt keine Veränderung. Das kann z.B. passieren, wenn die
Wirtspflanze das notwendige Substrat gar nicht zur Verfügung stellt. Eine
gemeine Variante von Pech: Das 'richtige' Substrat ist nicht da, aber andere
ebenfalls akzeptierte Substanzen,
und dann kann die Promiskuität der Enzyme dazu führen, dass Sie etwas ganz
Unerwartetes Neues bekommen, das mit der eigentlichen Funktion in der
Ursprungspflanze gar nichts zu tun hat.
Wenn alles fehlschlägt, die neue Funktion eindeutig zu identifizieren, und Sie
möchten trotzdem publizieren: Beten Sie um einen Reviewer, der die Problematik
kennt und akzeptiert. Allerdings sollte man schon demonstrieren können,
dass man das Mögliche versucht hat.....
Zum Schluss noch eine Anmerkung. Heutzutage liefern
Genomprojekte eine grosse Zahl von Sequenzen, und in den meisten Fällen tauchen
dabei auch Typ III PKS auf. Das kann sehr interessant sein, da hier eine Chance
zur Entdeckung neuer Funktionen besteht. Allerdings sollte man hier auch zur
Vorsicht raten. Wenn man überhaupt keine Idee zur physiologischen Funktion hat,
d.h. wenn kein Naturstoff bekannt ist, der über eine Typ III PKS synthetisiert
werden könnte, dann sollte man sich gut überlegen, ob man mit solchen Genen
arbeiten sollte: Die bekannte Promiskuität wird ganz sicher dazu führen, dass
ein rekombinantes Enzym in vitro irgendein Substrat akzeptiert und irgend etwas
damit synthetisiert. Aber wie interpretiert man ein solches Ergebnis? Man sollte dann schon
parallel eine umfassende Analyse der Naturstoffe in der Pflanzen durchführen, aber wieviele
Arbeitsgruppen haben die Möglichkeiten dazu, und viel wichtiger, die Motivation, wenn
vorher überhaupt nichts bekannt ist?
Zum Seitenanfang
4.
Typ
III PKS; physiologische Rolle
unbekannt oder unsicher
-> Eine passende
Bezeichnung: 'Orphan PKS' (deutsch, aber nicht sonderlich gut:
'Waisen-PKS')
Zum Seitenanfang
Zitate
-
Abe, I., Sano, Y., Takahashi,
Y., Noguchi, H., 2003. Site-directed mutagenesis of
benzalacetone synthase: the role of Phe215 in plant type III polyketide
synthases. Journal of Biological Chemistry 278, 25218-25226.
Benzalacetone synthase (BAS) and chalcone synthase (CHS) are plant-specific
type III polyketide synthases (PKSs) that share [~]70% amino acid sequence
identity. BAS catalyzes a one-step decarboxylative condensation of
4-coumaroyl-CoA with malonyl-CoA to produce a diketide benzalacetone,
whereas CHS performs sequential condensations with three malonyl-CoA to
generate a tetraketide chalcone. A homology model suggested that BAS has the
same overall fold as CHS with cavity volume almost as large as that of CHS.
One of the most characteristic features is that Rheum palmatum BAS lacks
active site Phe-215; the residues 214LF conserved in type III PKSs are
uniquely replaced by IL. Our observation that the BAS I214L/L215F mutant
exhibited chalcone-forming activity in a pH-dependent manner supported a
hypothesis that the absence of Phe-215 in BAS accounts for the interruption
of the polyketide chain elongation at the diketide stage. On the other hand,
Phe-215 mutants of Scutellaria baicalensis CHS (L214I/F215L, F215W, F215Y,
F215S, F215A, F215H, and F215C) afforded increased levels of truncated
products; however, none of them generated benzalacetone. These results
confirmed the critical role of Phe-215 in the polyketide formation reactions
and provided structural basis for understanding the structure-function
relationship of the plant type III PKSs.
Zurück
-
Abe,
I., Takahashi, Y., Lou, W., Noguchi, H., 2003. Enzymatic formation of
unnatural novel polyketides from alternate starter and nonphysiological
extension substrate by chalcone synthase. Organic Letters 5, 1277-1280.
In the
chalcone synthase (CHS) enzyme reaction, both the starter molecule and the
extension unit of the poyketide chain elongation reaction were
simultaneously replaced with nonphysiological substrates. When incubated
with benzoyl-CoA and methylmalonyl-CoA as substrates, recombinant CHS from
Scutellaria baicalensis afforded an unnatural novel triketide,
4-hydroxy-3,5-dimethyl-6-phenyl-pyran-2-one, along with a tetraketide,
4-hydroxy-3,5-dimethyl-6-(1-methyl-2-oxo-2-phenyl-ethyl)-pyran-2-one. On the
other hand, the enzyme also accepted hexanoyl-CoA and methylmalonyl-CoA as
substrates to produce an unnatural novel triketide,
4-hydroxy-3,5-dimethyl-6-pentyl-pyran-2-one.
Zurück zum Text
-
Abe,
I., Takahashi, Y., Noguchi, H., 2002. Enzymatic formation of an unnatural C6-C5
aromatic polyketide by plant type III polyketide synthases. Organic Letters 4,
3623-3626.
Substrate specificities of plant polyketide synthases (PKSs) were
investigated using analogues of malonyl-CoA, the extension unit of the
polyketide chain elongation reactions. When incubated with methylmalonyl-CoA
and 4-coumaroyl-CoA, plant PKSs (chalcone synthase from Scutellaria
baicalensis, stilbene synthase from Arachis hypogaea, and
benzalacetone synthase from Rheum palmatum) afforded an unnatural
C(6)-C(5) aromatic polyketide, 1-(4-hydroxyphenyl)pent-1-en-3-one, formed by
one-step decarboxylative condensation of the two substrates. In contrast,
succinyl-CoA was not accepted as a substrate by the enzymes.
Zurück
-
Abe,
T., Noma, H., Noguchi, H., Abe, I., 2006. Enzymatic formation of an unnatural
methylated triketide by plant type III polyketide synthases.
Tetrahedron Letters 47, 8727-8730.
Octaketide synthase, a novel plant-specific type III polyketide
synthase from Aloe arborescens, efficiently accepted (2RS)-methylmalonyl-CoA
as a sole substrate to produce 6-ethyl-4-hydroxy-3,5-dimethyl-2-pyrone. On
the other hand, a tetraketide-producing chalcone synthase from Scutellaria
baicalensis and a diketide-producing benzalacetone synthase from Rheum
palmatum also yielded the unnatural methylated C9 triketide pyrone as a
single product by sequential decarboxylative condensations of three
molecules of (2RS)-methylmalonyl-CoA.
Zurück
-
Abe, I.,
Takahashi, Y., Morita, H., Noguchi, H., 2001. Benzalacetone synthase: a novel
polyketide synthase that plays a crucial role in the biosynthesis of
phenylbutanones in Rheum palmatum. European Journal of Biochemistry
268, 3354-3359.
Benzalacetone synthase (BAS) is a novel plant-specific polyketide synthase
that catalyzes a one step decarboxylative condensation of 4-coumaroyl-CoA
with malonyl-CoA to produce the C 6 -C 4 skeleton of phenylbutanoids in
higher plants. A cDNA encoding BAS was for the first time cloned and
sequenced from rhubarb (Rheum palmatum), a medicinal plant rich in
phenylbutanoids including pharmaceutically important phenylbutanone
glucoside, lindleyin (anti-inflammatory action in extracts, Fig. 4:
derivative of raspberry ketone, by attaching a sugar+phenyl-derivative to
hydroxy group of coumaroyl starter residue). The cDNA encoded a 42 kDa
protein that shares 60-75% amino acid sequence identity with other members
of the CHS-superfamily enzymes. Interestingly, R. palmatum BAS lacks
the active-site Phe215 residue (numbering in CHS) which has been proposed to
help orient substrates and intermediates during the sequential condensation
of 4-coumaroyl-CoA with malonyl-CoA in CHS. On the other hand, the catalytic
cysteine-histidine dyad (Cys164 - His303) in CHS is well conserved in BAS. A
recombinant enzyme expressed in E. coli efficiently afforded
benzalacetone as a single product from 4-coumaroyl-CoA and malonyl-CoA.
Further, in contrast with CHS that showed broad substrate specificity toward
aliphatic CoA esters, BAS did not accept hexanoyl-CoA, isobutyryl-CoA,
isovaleryl-CoA, and acetyl-CoA as a substrate. Finally, besides the
phenylbutanones in rhubarb, BAS has been proposed to play a crucial role for
the construction of the C 6 -C 4 moiety of a variety of natural products
such as medicinally important gingerols in ginger plant.
Zurück
-
Akiyama,
T., Shibuya, M., Liu, H. M., Ebizuka, Y., 1999. p-Coumaroyltriacetic
acid synthase, a new homologue of chalcone synthase, from Hydrangea
macrophylla var. thunbergii. European Journal of Biochemistry 263,
834-839.
Chalcone synthase and stilbene synthase are plant-specific polyketide
synthases. They catalyze three common consecutive decarboxylative
condensations and specific cyclization reactions. They are highly homologous
to each other, and are likely to fall into a family of polyketide synthases
along with acridone synthase and bibenzyl synthase. Two cDNA clones (named
HmC and HmS), both of which show high homology to the known chalcone
synthases, were obtained from leaves of Hydrangea macrophylla
var. thunbergii. They were expressed in Escherichia coli in
order to determine their enzyme functions. Detection of chalcone formation
clearly indicated that HmC encoded chalcone synthase, while HmS protein
catalyzed the formation of neither chalcone nor stilbene. However, a novel
pyrone, a lactonization product of a linear tetraketide was detected in
reaction products of HmS protein. This proves that HmS encodes a novel
polyketide synthase that catalyzes only chain elongation without cyclization.
Zurück
-
Beckert,
C., Horn, C., Schnitzler, J.-P., Lehning, A., Heller, W., Veit, M., 1997.
Styrylpyrone biosynthesis in Equisetum arvense L. Phytochemistry
44, 275-283.
Styrylpyrone synthase was detected in cell free extracts from gametophytes
of Equisetum arvense. This new enzyme catalyses the formation of
styrylpyrones from malonyl-CoA and hydroxycinnamoyl- CoA precursors. A
standard enzyme assay was established. The enzyme activity was characterized
in partially purified protein extracts. p-Coumaroyl-CoA was accepted as
substrate at pH 6.0-8.5 in various buffer systems with the formation of
bisnoryangonin, and optimum enzyme activity was observed in potassium
phosphate buffer at pH 7.5. Caffeoyl-CoA was accepted as substrate only in
potassium phosphate buffer at pH 6.0-7.5 with formation of hispidin; optimum
enzyme activity was observed at pH 7.0. The apparent K-m values were 220 µM
for caffeoyl-CoA and 230 µM for p-coumaroyl-CoA. The temperature optimum of
the enzyme activity was 37 degree for bisnoryangonin and 30 degree for
hispidin formation. Molecular weight determination by FPLC indicated that
this protein has a native molecular weight of ca 56-77 kDa. Styrylpyrones
accumulate in rhizomes of sporophytes and gametophytes of E. arvense
as major constitutive metabolites. In these organs no flavonoids could be
detected. In green sprouts, styrylpyrone accumulation is only detected as a
local response to mechanical wounding or microbial attack, and flavonoids
are accumulated as major polyketide metabolites. Thus, chalcone synthase is
active in the sporophytes and might have developed in the course of
evolution from styrylpyrone synthase present in the more primitive
gametophytes.
Zurück
-
Borejsza-Wysocki,
W., Hrazdina, G., 1994. Biosynthesis of p-hydroxyphenylbutan-2-one in
raspberry fruits and tissue cultures. Phytochemistry 35, 623-628.
p-Hydroxyphenylbutan-2-one (pHPB), the raspberry ketone, is responsible for
the characteristic aroma of raspberries. The compound accumulates rapidly
during the later maturation stages of the berries. The synthesis and
accumulation of pHPB correlates with that of anthocyanin and soluble solids
( degree Brix). pHPB is synthesized in cell-free extracts of fruits and
tissue cultures from p-coumaryl-CoA and malonyl-CoA in a manner similar to
the synthesis of chalcones and stilbenes. The specific biosynthetic pathway
for pHPB formation deviates from the general phenylpropanoid pathway at the
p-coumaryl-CoA stage and it is composed of two enzymes. The first enzyme is
the p-hydroxyphenylbut-3-ene-2-one synthase (pHPB-3-ene-2-one synthase) that
forms p-hydroxyphenylbut-3-ene-2-one by the condensation of malonyl-CoA with
p-coumaryl-CoA. The second enzyme, p- hydroxyphenylbut-3-ene-2-one reductase
(pHPB-3-ene-2-one reductase), reduces the p-hydroxyphenylbut-3-ene-2-one to
p-hydroxyphenylbutan-2-one, the raspberry ketone. We detected the activity
of both enzymes in crude extracts from raspberry fruits and their tissue
cultures, and identified their reaction products by HPLC, crystallization to
constant radioactivity and by GC-MS.
Zurück
-
Borejsza-Wysocki,
W., Hrazdina, G., 1996. Aromatic polyketide synthases. Purification,
characterization, and antibody development to benzalacetone synthase from
raspberry fruits. Plant Physiology 110, 791-799.
p-Hydroxyphenylbutan-2-one, the characteristic aroma compound of raspberries
(Rubus idaeus L.), is synthesized from p-coumaryl-coenzyme A and
malonyl-coenzyme A in a two-step reaction sequence that is catalyzed by
benzalacetone synthase and benzalacetone reductase (W. Borejsza-Wysocki and
G. Hrazdina 1994, Phytochemistry 35: 623-628). Benzalacetone synthase
condenses one malonate with p-coumarate to form the pathway intermediate p-
hydroxyphenylbut-3-ene-2-one (p-hydroxybenzalacetone) in a reaction that is
similar to those catalyzed by chalcone and stilbene synthases. We have
obtained an enzyme preparation from ripe raspberries that was preferentially
enriched in benzalacetone synthase (approximately 170-fold) over chalcone
synthase (approximately 14-fold) activity. This preparation was used to
characterize benzalacetone synthase and to develop polyclonal antibodies in
rabbits. Benzalacetone synthase showed similarity in its molecular
properties to chalcone synthase but differed distinctly in its substrate
specificity, response to 2-mercaptoethanol and ethylene glycol, and
induction in cell- suspension cultures. The product of the enzyme,
p-hydroxybenzalacetone, inhibited mycelial growth of the raspberry pathogen
Phytophthora fragariae var rubi at 250 µM. We do not know whether the
dual activity in the benzalacetone synthase preparation is the result of a
bifunctional enzyme or is caused by contamination with chalcone synthase
that was also present. The rapid induction of the enzyme in cell-suspension
cultures upon addition of yeast extract and the toxicity of its product,
p-hydroxybenzalacetone, to phytopathogenic fungi also suggest that the
pathway may be part of a plant defense response.
Zurück
-
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.
Anmerkung:
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
(mehr...).
Zurück
-
Hrazdina, G., Zheng, D. S., 2006.
Expression and function of aromatic polyketide synthase genes in raspberries (Rubus
idaeus sp.). In: Rimando, A. M., Baerson, S. R. (Eds.), Polyketides:
Biosynthesis, Biological Activities and Genetic Engineering, American Chemical
Society, Washington, D.C., pp. 128-140.
The genus
Rubus contains a family of type III polyketide synthases which show
differences in structure and function. We have cloned and characterized five
aromatic polyketide synthase genes from raspberries. All five genes contain
an intron of varying length and have 1173 bp coding sequences, with the
exception of one gene that consists of 1149 bp. Four of the five genes
encode proteins with 391 amino acid residues with a calculated protein mass
of 42 kDa, while one gene coded for a shorter protein consisting of 383
amino acids. Sequence comparison of the five polyketide synthase genes
showed high similarity both at the DNA and protein levels. Differences in
the coding region were found mainly in the flanking sequences. Analysis of
the reaction products showed that PKS1 and PKS5 were chalcone synthases,
PKS2 that differs in six amino acids from PKS1 is silent, PKS3 is a p-coumarate
triacetic acid lactone synthase (CTAS) and PKS4 is a benzalacetone synthase
(BAS). The structural variations and the architecture of these PKS genes and
enzymes is discussed.
Zurück
-
Jiang, C., Kim, S. Y., Suh, D.-Y., 2008. Divergent
evolution of the thiolase superfamily and chalcone synthase family.
Molecular Phylogenetics and Evolution 49, 691-701.
Enzymes of the
thiolase superfamily catalyze the formation of carbon-carbon bond via the
Claisen condensation reaction. Thiolases catalyze the reversible
non-decarboxylative condensation of acetoacetyl-CoA from two molecules of
acetyl-CoA, and possess a conserved Cys-His catalytic diad. Elongation
enzymes (beta-ketoacyl-acyl carrier protein synthase (KAS) I and KAS II and
the condensing domain of polyketide synthase) have invariant Cys and two His
residues (CHH triad), while a Cys-His-Asn (CHN) triad is found in initiation
enzymes (KAS III, 3-ketoacyl-CoA synthase (KCS) and the chalcone synthase (CHS)
family). These enzymes all catalyze decarboxylative condensation reactions.
3-Hydroxyl-3-methylglutaryl-CoA synthase (HMGS) also contains the CHN triad,
although it catalyzes a non-decarboxylative condensation. That the enzymes
of the thiolase superfamily share overall similarity in protein structure
and function suggested a common evolutionary origin. All thiolases were
found to have, in addition to the Cys-His diad, either Asn or His (thus C(N/H)H)
at a position corresponding to the His in the CHH and CHN triads. In our
phylogenetic analyses, the thiolase superfamily was divided into four main
clusters according to active site architecture. During the functional
divergence of the superfamily, the active architecture was suggested to
evolve from the CHH in archaeal thiolases to the C(N/H)H in non-archaeal
thiolases, and subsequently to the CHH in the elongation enzymes and the CHN
in the initiation enzymes. Based on these observations and available
biochemical and structural evidences, a plausible evolutionary history for
the thiolase superfamily is proposed that includes the emergence of
decarboxylative condensing enzymes accompanied by a recruitment of the His
in the CHH and CHN triads for a catalytic role during decarboxylative
condensation. In addition, phylogenetic analysis of the plant CHS family
showed separate clustering of CHS and non-CHS members of the family with a
few exceptions, suggesting repeated gene birth-and-death and re-invention of
non-CHS functions throughout the evolution of angiosperms. Based on these
observations, predictions on the enzymatic functions are made for several
members of the CHS family whose functions are yet to be characterized.
Further, a moss CHS-like enzyme that is functionally similar to a
cyanobacterial enzyme was identified as the most recent common ancestor to
the plant CHS family.
Zurück
-
Kreuzaler, F., Hahlbrock, K., 1975a. Enzymatic synthesis of aromatic
compounds in higher plants. Formation of bis-noryangonin
(4-hydroxy-6[4-hydroxystyryl]2-pyrone) from p-coumaroyl-CoA and malonyl-CoA.
Archives of Biochemistry and Biophysics 169, 84-90.
Cell-free extracts from light-induced cell suspension cultures of
Petroselinum hortense catalyzed, in
the presence of mercaptoethanol or dithioerythritol, the formation of
bisnoryangonin from p-coumaroyl-CoA
and malonyl-CoA. Radioactivity from the 3H- and 14C-labeled
acyl moieties of p-coumaroyl-CoA
and malonyl-CoA, respectively, was incorporated into the product at a molar
ratio of 1:2. This result supports earlier conclusions from experiments
in vivo favoring a mechanism of
synthesis for the pyrone ring of bisnoryangonin according to the “acetate
rule.” Bis-noryangonin could not be detected in cultured
Petroselinum hortense cells
in vivo. Our present results
suggest that the styrylpyrone derivative formed
in vitro is an artificial product
of the first enzyme of the flavonoid pathway, flavanone synthetase. In the
course of a 300-fold purification of this enzyme, the
bis-noryangonin-synthesizing activity was always associated with the
flavanone synthetase activity. The concentration of certain thiol reagents,
such as mercaptoethanol or dithioerythritol, the ionic strength of the
buffer, and the degree of purity of the enzyme preparation had a pronounced,
differential effect on the amounts of flavanone and styrylpyrone formed by
the flavanone synthetase. A possible explanation for the mechanism of
formation of the artificial product, bis-noryangonin, is discussed.
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...).
Zurück
-
Kreuzaler, F., Hahlbrock, K., 1975b. Enzymic synthesis of an aromatic ring
from acetate units. Partial purification and properties of flavanone synthase
from cell-suspension cultures of Petroselinum hortense. European
Journal of Biochemistry 56, 205-213.
Flavanone synthase was isolated and purified about 300-fold from
fermenter-grown, light-induced cell suspension cultures of Petroselinum
hortense. The enzyme catalyzed the formation of the flavanone naringenin
from p-coumaroyl-CoA and malonyl-CoA. Trapping experiments with an enzyme
preparation, which was free of chalcone isomerase activity, revealed that in
fact the flavanone and not the isomeric chalcone was the immediate product
of the synthase reaction. Thus the enzyme is not a chalcone synthase as
previously assumed. No coafactors were required for flavanone synthase
activity. The enzyme was strongly inhibited by the two reaction products
naringenin and CoASH, by the antibiotic cerulenin, by acetyl-CoA, and by
several compounds reacting with sulfhydryl groups. Optimal enzyme activity
was found at pH 8.0, at 30 degrees C, and at an ionic strength of 0.1 - 0.3
M potassium phosphate. EDTA, Mg2+, Ca2+, or Fe2+ at concentrations of about
0.7 muM did not affect the enzyme activity. Apparent molecular weights of
approx. 120 000, 50 000, and 70 000, respectively, were determined for
flavanone synthase and two metabolically related enzymes, chalcone isomerase
and malonyl-CoA: flavonoid glycoside malonyl transferase. The partially
purified flavanone synthase efficiently catalyzed the formation of malonyl
pantetheine from malonyl-CoA and pantetheine. This malonyl transferase
activity, and a general similarity with the condensation steps involved in
the mechanisms of fatty acid and 6- methylsalicylic acid synthesis from "acetate
units", are the basis for a hypothetical scheme which is proposed for the
sequence of reactions catalyzed by the multifunctional flavanone synthase.
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
(mehr...).
Zurück
-
Morita, H.,
Noguchi, H., Schröder, J., Abe, I., 2001. Novel polyketides
synthesized with a higher plant stilbene synthase. European Journal of
Biochemistry 268, 3759-3766.
The
physiological function of the stilbene synthase (STS) from groundnut (Arachis
hypogaea) is the formation of resveratrol. The enzyme uses
4-coumaroyl-CoA, performs three condensations with malonyl-CoA, and folds
the resulting tetraketide into a new aromatic ring system. We investigated
the capacity to build novel and unusual polyketides from alternative
substrates. Three types of products were obtained: (A) complete reaction (stilbene-type),
(B) three condensations without formation of aromatic ring (CTAL-type pyrone
derailment), (C) two condensations (BNY-type pyrone derailment). All product
types were obtained from 4-fluorocinnamoyl-CoA and analogs in which the
coumaroyl moiety was replaced by furan or thiophene. Only type (B) and (C)
products were synthesized from other 4-substituted 4-coumaroyl-CoA analogs
(-Cl, -Br, -OCH3). Benzoyl-CoA, phenylacetyl-CoA, and medium
chain aliphatic CoA-esters were poor substrates, and the majority of the
products was of type (C). The results show that minor modifications can be
used to direct the enzyme reaction to form a variety of different and new
products. Manipulation of the biosynthesis of polyketides by synthetic
analogs could lead to development of a chemical library of pharmaceutically
interesting novel polyketides.
Zurück
Anfrage für Sonderdruck
-
Morita, H., Takahashi, Y., Noguchi, H., Abe, I., 2000. Enzymatic formation
of unnatural aromatic polyketides by chalcone synthase. Biochemical and
Biophysical Research Communications 279, 190-195.
Substrate specificity of recombinant chalcone synthase (CHS) from
Scutellaria baicalensis (Labiatae) was investigated using chemically
synthesized aromatic and aliphatic CoA esters. It was demonstrated for the
first time that CHS converted benzoyl-CoA to phlorobenzophenone
(2,4,6-trihydroxybenzophenone) along with pyrone by-products. On the other
hand, phenylacetyl-CoA was enzymatically converted to an unnatural aromatic
polyketide, phlorobenzylketone (2,4,6-trihydroxyphenylbenzylketone), whose
structure was finally confirmed by chemical synthesis. Furthermore, in
agreement with earlier reports, S. baicalensis CHS also accepted
aliphatic CoA esters, isovaleryl-CoA and isobutyryl-CoA, to produce
phloroacylphenones. In contrast, hexanoyl-CoA only afforded pyrone
derivatives without formation of a new aromatic ring. It was noteworthy that
both aromatic and aliphatic CoA esters were accepted in the active site of
the enzyme as a starter substrate for the complex condensation reaction. The
low substrate specificity of CHS thus provided further insight into the
structure and function of the enzyme.
Zurück to text
-
Saleh,
N. A. M., Fritsch, H., Kreuzaler, F., Grisebach, H., 1978. Flavanone synthase
from cell suspension cultures of Haplopappus gracilis and
comparison with the synthase from parsley. Phytochemistry 17, 183-186.
Flavanone synthase was isolated and purified
ca 62-fold from cell suspension
cultures of Haplopappus gracilis.
The enzyme preparation catalysed the formation of naringenin from
4-coumaryl-CoA and malonyl-CoA with a pH optimum of
ca 8. The same enzyme was also
capable of synthesizing eriodictyol from caffeyl-CoA and malonyl-CoA; in
this case the pH optimum lay between 6.5 and 7. The homogeneous flavanone
synthase from cell suspension cultures of parsley showed the same dependence
of the pH optimum on the nature of the cinnamyl-CoA. It can be concluded
that both naringenin and eriodictyol are natural products of the synthase
reaction.
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: more...
Zurück
-
Schröder, J., Raiber, S., Berger, T., Schmidt, A.,
Schmidt, J., Soares-Sello, A. M., Bardshiri, E., Strack, D., Simpson, T. J.,
Veit, M., Schröder, G., 1998. Plant polyketide synthases: a chalcone
synthase-type enzyme which performs a condensation reaction with
methylmalonyl-CoA in the biosynthesis of C-methylated chalcones. Biochemistry
37, 8417-8425.
Heterologous screening of a cDNA library from Pinus
strobus seedlings identified clones for two chalcone synthase (CHS)
related proteins (PStrCHS1 and PStrCHS2, 87.6% identity). Heterologous
expression in Escherichia coli showed that PStrCHS1 performed
the typical CHS reaction, that it used starter CoA-esters from the
phenylpropanoid pathway, and that it performed three condensation reactions
with malonyl-CoA, followed by the ring closure to the chalcone. PstrCHS2 was
completely inactive with these starters and also with linear CoA-esters.
Activity was detected only with a diketide derivative (N-acetylcysteamine
thioester of 3-oxo-5-phenylpent-4-enoic acid) that corresponded to the CHS
reaction intermediate postulated after the first condensation reaction.
PstrCHS2 performed only one condensation, with 6-styryl-4-hydroxy-2-pyrone
derivatives as release products. The enzyme preferred methylmalonyl-CoA
against malonyl-CoA, if only methylmalonyl-CoA was available. These
properties and a comparison with the CHS from Pinus sylvestris
suggested for PstrCHS2 a special function in the biosynthesis of secondary
products. In contrast to P. sylvestris, P. strobus
contains C-methylated chalcone derivatives, and the methyl group is at the
position predicted from a chain extension with methylmalonyl-CoA in the
second condensation of the biosynthetic reaction sequence. We propose that
PstrCHS2 specifically contributes the condensing reaction with
methylmalonyl-CoA to yield a methylated triketide intermediate. We discuss a
model that the biosynthesis of C-methylated chalcones represents the
simplest example of a modular polyketide synthase.
Sonderdruckanfrage
Zurück zum Text oder springen Sie zu den
C-methylierten Flavonoiden
-
Schröder,
J., 1999. The chalcone/stilbene synthase-type family of condensing enzymes.
In: Sankawa, U. (Ed.), Polyketides and Other Secondary Metabolites Including
Fatty Acids and Their Derivatives, Vol. 1.
Elsevier, Amsterdam, pp. 749-771.
Sonderdruckanfrage
Zurück
-
Schüz, R., Heller, W., Hahlbrock, K., 1983. Substrate specificity of
chalcone synthase from Petroselinum hortense. Formation of
phloroglucinol derivatives from aliphatic substrates. Journal of Biological
Chemistry 258, 6730-6734.
The substrate specificity of chalcone synthase, the key enzyme of flavonoid
biosynthesis, was investigated. A purified enzyme preparation from cell
suspension cultures of parsley (P. hortense) catalyzed chain
elongations with acetate units from malonyl-CoA, using various aromatic and
aliphatic CoA esters as starter molecules. Malonyl-CoA could not be replaced
by malonyl acyl carrier protein in the standard chalcone synthase assay.
Butyryl-CoA, hexanoyl-CoA and benzoyl-CoA served as substrates for the
condensation reaction with similar efficiency as 4- coumaroyl-CoA, the
natural substrate of the enzyme. Acetyl-CoA and octanoyl-CoA were relatively
poor substrates. Among the products formed with the 2 most efficient
aliphatic substrates tested, butyryl-CoA and hexanoyl-CoA, were the
respective chalcone analogs, phlorobutyrophenone and phlorocaprophenone.
Chalcone synthase and the corresponding enzyme of fatty acid synthesis in
higher plants, beta-ketoacyl-acyl carrier protein synthase, may have a
common evolutionary origin.
Zurück
-
Suh,
D.-Y., Kagami, J., Fukuma, K., Iwanami, N., Yamazaki, Y., Yurimoto, H.,
Sakai, Y., Kato, N., Shibuya, M., Ebizuka, Y., Sankawa, U., 2000. Chalcone and
stilbene synthases expressed in eucaryotes exhibit reduced cross-reactivity in vitro. Chemical & Pharmaceutical Bulletin 48, 1051-1054.
Chalcone synthase (CHS) and stilbene synthase (STS) catalyze different
cyclization reactions of the common tetraketide to give different products,
naringenin chalcone and resveratrol, respectively. We have previously
observed in vitro cross-reaction of CHS and STS overexpressed in
Escherichia coli, resveratrol production by CHS and chalcone production
by STS. When expressed in eucaryotic cells, or in E. coli as
thioredoxin-fusion proteins, CHS and STS exhibited reduced cross-reaction.
STS refolded from inclusion bodies also showed reduced cross-reaction. While
addition of bovine serum albumin and pH in the reaction were without
noticeable effect, addition of glycerol decreased the cross-reaction of CHS
likely due to its stabilizing effect on enzyme conformation. These results
were interpreted to provide supporting evidence to our earlier proposition (Yamaguchi
T. et al., FEBS Lett., 460, 457-461 (1999)) that the in vitro cross-
reaction of CHS and STS is due to intrinsic capability of these enzymes to
catalyze different types of cyclization, which, in turn, is endowed by
conformational flexibility of their active sites.
Zurück zum Text
-
Yamaguchi, T., Kurosaki, F., Suh, D.-Y., Sankawa, U., Nishioka, M.,
Akiyama, T., Shibuya, M., Ebizuka, Y., 1999. Cross-reaction of chalcone
synthase and stilbene synthase overexpressed in Escherichia coli.
FEBS Letters 460, 457-461.
Chalcone synthase (CHS) and
stilbene synthase (STS) are related plant polyketide synthases belonging to
the CHS superfamily. CHS and STS catalyze common condensation reactions of
p-coumaroyl-CoA and three C-2-units from malonyl-CoA but different
cyclization reactions to produce naringenin chalcone and resveratrol,
respectively. Using purified Pueraria lobata CHS and Arachis
hypogaea STS overexpressed in Escherichia coli, bisnoryangonin (BNY,
the derailed lactone after two condensations) and p-coumaroyltriacetic acid
lactone (the derailed lactone after three condensations) were detected from
the reaction products. More importantly, we found a crossreaction between
CHS and STS, i.e. resveratrol production by CHS (2.7-4.2% of naringenin) and
naringenin production by STS (1.4-2.3% of resveratrol), possibly due to the
conformational flexibility of their active sites.
Zurück zum Text
-
Yu, C. K. Y., Springob, K., Schmidt, J., Nicholson, R.
L., Chu, I. K., Yip, W. K., Lo, C., 2005. A stilbene synthase gene (SbSTS1)
is involved in host and non-host defense responses in Sorghum.
Plant Physiology 138, 393-401.
A chalcone
synthase (CHS)-like gene, SbCHS8, with high expressed sequence tag
abundance in a pathogen-induced cDNA library, was identified previously in
sorghum (Sorghum bicolor). Genomic Southern analysis revealed that
SbCHS8 represents a single-copy gene. SbCHS8 expression was
induced in sorghum mesocotyls following inoculation with Cochliobolus
heterotrophus and Colletotrichum sublineolum, corresponding to
nonhost and host defense responses, respectively. However, the induction was
delayed by approximately 24 h when compared to the expression of at least
one of the other SbCHS genes. In addition, SbCHS8 expression
was not induced by light and did not occur in a tissue-specific manner.
SbCHS8, together with SbCHS2, was overexpressed in transgenic
Arabidopsis (Arabidopsis thaliana) tt4 (transparent testa)
mutants defective in CHS activities. SbCHS2 rescued the ability of
these mutants to accumulate flavonoids in seed coats and seedlings. In
contrast, SbCHS8 failed to complement the mutation, suggesting that
the encoded enzyme does not function as a CHS. To elucidate their
biochemical functions, recombinant proteins were assayed with different
phenylpropanoid-Coenzyme A esters. Flavanones and stilbenes were detected in
the reaction products of SbCHS2 and SbCHS8, respectively. Taken together,
our data demonstrated that SbCHS2 encodes a typical CHS that
synthesizes naringenin chalcone, which is necessary for the formation of
different flavonoid metabolites. On the other hand, SbCHS8, now
retermed SbSTS1, encodes an enzyme with stilbene synthase activity,
suggesting that sorghum accumulates stilbene-derived defense metabolites in
addition to the well-characterized 3-deoxyanthocyanidin phytoalexins.
Zurück zum Text
-
Zheng, D., Hrazdina, G., 2008.
Molecular and
biochemical characterization of benzalacetone synthase and chalcone synthase
genes and their proteins from raspberry (Rubus idaeus L). Archives of
Biochemistry and Biophysics 470,
139-145.
Two new members of the polyketide synthase (PKS) gene family (RiPKS4
and RiPKS5) were cloned from raspberry fruits (Rubus idaeus L.,
cv Royalty) and expressed in E. coli. Characterization of the
recombinant enzyme products indicated that RiPKS4 is a
bifunctional polyketide synthase producing both 4-hydroxybenzalacetone and
naringenin chalcone. The recombinant RiPKS4 protein, like the native
protein from raspberry fruits accepted p-coumaryl-CoA and
ferulyl-CoA as starter substrates and catalyzed the formation of both
naringenin chalcone, 4-hydroxy-benzalacetone and
3-methoxy-4-hydroxy-benzalacetone. Although activity of RiPKS4 was
higher with ferulyl-CoA than with p-coumaryl-CoA, the corresponding
product, 3-methoxy-4-hydroxy phenylbutanone could not be detected in
raspberries to date. Sequence analysis of the genes and proteins suggested
that this feature of RiPKS4 was created by variation in the C-terminus
due to DNA recombination at the 3'region of its coding sequence. RiPKS5
is a typical chalcone synthase (CHS) that uses p-coumaryl-CoA only as
starter substrate and produces naringenin chalcone exclusively as the reaction
product.
Zurück zum Text
Zum
Seitenanfang
File History:
.
|
