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(Last modification:
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Valerophenonsynthase (VPS)
Humulus lupulus (hop, Hopfen, gehört zu den Hanfgewächsen, Cannabaceae)
wird seit mehreren Jahrhunderten in der Herstellung von Bier verwendet. Der
ursprüngliche Grund war nicht der Geschmack, wie häufig angenommen, sondern die
stabilisierende Wirkung durch die Bittersäuren in den Fruchtständen. Hopfen
hatte sich dann etwa im 13. Jahrhundert als das Beste durchgesetzt. In England
jedoch dauerte es weitere 200 Jahre bis Hopfen als die beste Wahl akzeptiert
wurde; davor wurde er anscheinend als 'wicked and pernicious weed' verunglimpft
(etwa so: bösartiges und hartnäckiges Unkraut). Das Bayerische Reinheitsgebot
von 1516 legte dann fest, dass in Deutschland nur Hopfen verwendet werden
durfte. Eine interessante Zusammenfassung findet sich in einem 'Millenium
Review' (Moir, 2000). Wikipedia
erzählt auch etwas über Hopfen, und führt auch zu einigen guten Bildern. Der
deutsche Artikel ist besser als der englische, aber in beiden Fällen lässt
die Beschreibung der Naturstoffe viel zu wünschen übrig. Humulon und Lupulon,
die beiden uns hier interessierenden Naturstoffe, werden gar nicht besprochen
(Vorsicht, bitte aufpassen: Nicht Humulon
mit dem dort erwähnten Humulen
verwechseln. Humulen
ist ein Sesquiterpen, und wird nicht über eine PKS-Reaktion gebildet!).
Humulon und Lupulon sind die vorherrschenden
Bitterstoffe in den Fruchtständen, und bereits 1994 wurde vorgeschlagen, dass
eine CHS-Typ Reaktion in der Biosynthese des Grundgerüstes beteiligt ist (ganz
schön clever: Würden Sie in Humulon das Wirken einer CHS-Typ Reaktion
erkennen?). Startersubstrate sollten Isovaleryl-CoA (-> Humulon) und
Isobutyryl-CoA (-> Lupulon) sein (Fung et al., 1994).
Bereits ein Jahr danach wurde die vorgesagte Enzymaktivität in vitro
gezeigt(Zuurbier
et al., 1995), aber die völlige Reinigung des Enzyms und die
Mikrosequenzierung von Peptide dauerte etwas länger: Die Sequenzen zeigten klare
Verwandtschaft mit den damals bekannten Chalconsynthasen (CHS) (Paniego
et al., 1999). Das Enzym wurde dann als Valerophenonsynthase (VPS) bezeichnet,
nach dem vorzugsweise verwendeten Substrat. Die Abbildung zeigt das Prinzip der
Reaktion und das Endprodukt, Humulon.
Valerophenonsynthase (VPS) in
Hopfen (Humulus lupulus): eine Typ III PKS.
Die Farben
(●●●)
geben die Kondensationsreaktionen an und die C-Atome, die durch
die drei Kondensations-Reaktionen eingeführt werden.
Das hier gezeigte Beispiel ist mit Isovaleryl-CoA als Startersubstrat der PKS-Reaktion. Humulon ist das Endprodukt nach mehreren Modifikationen (gestrichelter
Pfeil). Lupulone ist das entsprechende Produkt aus Isobutyryl-CoA. Grün:
Prenylrest-Seitenketten.
Der Vorschlag für ein CHS-verwandtes Protein passte zu den
Ergebnissen mit einer echten CHS (aus der Kiefer, Pinus sylvestris) mit
Isovaleryl-CoA als Substrat: Die CHS konnte die vorgesagte Reaktion durchführen,
aber nicht perfekt, denn die Mehrheit der Produkte waren die Pyrone von nur zwei
Kondensations-Reaktionen (Zuurbier et al.,
1998).
Die erste VPS cDNA wurde in 2001 publiziert (Okada
and Ito, 2001), und wenig später wurde eine CHS cDNA beschrieben (Matousek
et al., 2002). Von den oben erwähnten Ergebnissen mit einer 'echten' CHS
hätte man raten können, was dann passierte: Offensichtlich war es nicht so
einfach, die beiden Aktivitäten klar zu unterscheiden. Denn nicht nur die CHS
hatte VPS-Aktivität, sondern die VPS konnte auch als CHS funktionieren, und so
wurde dann zu der Zeit vorgeschlagen, dass ein Enzym sowohl den
Weg zu den Bitterstoffen als auch zu den Chalconen (Flavonoide) bedient (Okada
et al., 2001). Eine spätere Untersuchung berichtete, dass zwei der fünf
analysierten Typ III PKS sowohl CHS als auch VPS-Aktivitäten in vitro
hatten. Es wurde dann vorgeschlagen, dass das Enzym mit der höheren
VPS-Aktivität hauptsächlich zu der Biosynthese der Bitterstoffe beiträgt,
während das Protein mit höherer CHS-Aktivität wohl das Hauptenzym für die
Flavonoid-Biosynthese ist (Okada
et al., 2004). In einem solchen Fall ist es wohl nicht gut möglich,
eine physiologische Rolle aus in vitro Experimenten abzuleiten; man
benötigt dafür ganz andere Ansätze.
Orphan VPS
In diesem Zusammenhang ist es bemerkenswert, dass cDNAs für Proteine mit
VPS-Aktivität auch aus anderen Pflanzen kloniert wurden. Eine ganze Serie von
Typ III PKS erhielt man aus der einfachsten vaskulären Pflanze
Psilotum nudum.
Die Untersuchungen mit rekombinanten Proteinen ergaben Enzyme mit CHS,
Stilbensynthase (STS), und VPS Aktivitäten (Yamazaki et
al., 2001). Die Bedeutung der STS und VPS Aktivitäten bleibt mysteriös, denn
in der Pflanze sind überhaupt keine Naturstoffe bekannt, die von solchen
Reaktionen stammen, oder abgeleitet werden können. Beim gegenwärtigen
Kenntniss-Stand sollten diese Enzyme als 'Orphan
PKS' bezeichnet werden, da die physiologische Rolle völlig unklar ist.
Ein anderes solches Beispiel
ist eine cDNA aus dem Hanf (Cannabis sativa). Das Protein hatte CHS und
VPS-Aktivität, und war 94% identisch mit einer der 'CHS' aus dem Hopfen
(Raharjo
et al., 2004). Die grosse Ähnlichkeit lässt sich vielleicht damit erklären,
dass Hanf und Hopfen der gleichen Familie angehören (Cannabaceae).
Zusammenfassend, wie bereits oben bemerkt: Enzymteste in vitro haben
offensichtlich nur sehr begrenzten Wert zur Definition der 'wirklichen'
Substrate, wenn diese praktisch von jeder beliebigen CHS akzeptiert werden
(mehr...)
.
Bereits 1983 wurde mit der CHS von Petersilie gezeigt, dass CoA-Ester von
kurzkettigen Fettsäuren excellente Substrate für CHS sind (
Schüz et al. 1983).
Links zu anderen Beispielen von 'Orphan PKS'
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Zitate
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Fung, S.-Y., Brussee, J., Van der Hoeven, R. A. M.,
Niessen, W. M. A., Scheffer, J. J. C., Verpoorte, R., 1994.
Analysis of proposed
aromatic precursors of hop bitter acids. Journal of Natural Products 57,
452-459.
Four proposed precursors ofhop
bitter acids, 2-acyl-1,3,5-benzenetriol and
2-acyl-4-(3-methyl-2-butenyl)-1,3,5-benzenetriol (acyl = methylpropanoyl
or 3-methylbutanoyl), were synthesized (compounds 7-10). TIC, hplc and
gc systems were developed for the analysis of these compounds in the
presence of the hop bitter acids 1-6. All four precursors were found to
be present in cone extracts of several hop cultivars.
Zurück zum Text
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Matousek, J., Novák, P., Bríza, J., Patzak, J.,
Niedermeierová, H., 2002.
Cloning and
characterisation of chs-specific DNA and cDNA sequences from hop (Humulus
lupulus L.). Plant Science 162, 1007-1018.
A
complete sequence of chalcone synthase (CHS) gene from hop was cloned.
The gene designated chs_H1 consists of two exons and one 187 bp
intron. CHS protein predicted from chs_H1 cDNA has 42.5 kDa and retains
conserved domains and residues including 26 amino acids at positions
identical to those identified by crystallography as characteristic for
catalytic domains of alfalfa CHS (EC 2.3.1.74). Cloned CHS_H1 protein
shows specific CHS activity with 4-coumaroyl-CoA. Structure modelling
revealed clear differences between CHS_H1 and phlorisovalerophenone
synthase, the only published CHS-like homologue from hop. Conserved
motifs like H, and G boxes characteristic for the light regulated and
stress inducible genes were identified within promoter region of chs_H1
gene. Highly specific expression of chs_H1 mRNA was detected by
quantitative RT PCR in glandular trichomes during cone maturation. Much
lower, but significant levels of chs_H1 mRNA were detected at the
stage of hop flowering in petioles (100%), developed flowers (96%), and
in stem apexes (78%), while the lowest levels of mRNA were found in the
roots (31%) and leaf blades (9%). Southern blot analyses predicted at
least five additional chs-like genes related to chs_H1. A
genomic arrangement different from phlorisovalerophenone synthase
sequences was found for these genes. RFLP analyses using DNA from 15
genotypes revealed several distinct dendrogram clusters, suggesting
specific re-arrangements of hop chs-like genes during evolution
and/or during the breeding and selection processes.
Zurück zum Text
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Moir, M., 2000. Hops - A millennium review. Journal
of the American Society of Brewing Chemists 58, 131-146.
Of all the
herbs that have been used to flavor and preserve beer over the ages,
only the hop (Humulus lupulus L.) is now regarded as an essential raw
material in brewing throughout the world. Although hops were cultivated
in Babylon as far back as 200 A.D., there is no record of their use to
make beer until about 1079. The value of hops for flavor and
preservation of alcoholic beverages appears to have been recognized by
the twelfth century, and, in the thirteenth century, the hop started to
threaten traditional herbs, such as rosemary, yarrow, coriander, and bog
myrtle. The Bavarian Purity Law of 1516 decreed that only hops could be
used for bittering beers in Germany. For a long time, sweet and strong
ales were preferred in England, and the hop was condemned as a 'wicked
and pernicious weed.' Good sense eventually prevailed, however, and hops
were first grown in England in 1524, 100 years before the first gardens
were established in North America. Although selection of improved
varieties must have been a continuous process for most of the second
millennium, the breeding of hops as an organized, scientific operation
dates from the start of the twentieth century with a program initiated
at Wye College, England, in 1904. In the same year, the alpha-acid
humulone was first isolated as a crystalline solid, and during the next
60 years our knowledge of hop chemistry and understanding of the
isomerization process was developed, leading to great benefits for
breeders, growers, and brewers alike. Over the last 40 years, there has
been a rapid proliferation of types of processed hops, or hop products,
which offer the brewer several economic and quality advantages over the
use of whole hops, and the contribution of hops to beer aroma,
bitterness, foam, and light stability can now be controlled more
effectively than ever before.
Zurück zum Text
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Okada, Y., Ito, K., 2001a. Cloning and analysis of
valerophenone synthase gene expressed specifically in lupulin gland of hop
(Humulus lupulus L.). Bioscience, Biotechnology and Biochemistry 65,
150-155.
Resin
and essential oil derived from hop (Humulus lupulus L.) cones are
very important compounds for beer brewing, and they specifically
accumulate in the lupulin gland of hop cones, In order to identify the
genes responsible for the biosynthetic pathway of these compounds and
use the identified genes for hop breeding using Marker Assisted
Selection and transformation techniques, genes expressed specifically in
the lupulin gland were cloned and sequenced. One of them was suggested
to be similar to the chalcone synthase gene from the DNA sequence. The
translation product of the gene had the activity of valerophenone
synthase, which catalyzes a part of the synthesis reaction of alpha
-acid and beta -acid, Northern analysis showed that the valerophenone
synthase gene seemed to be expressed specifically in the lupulin gland.
Zurück zum Text
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Okada, Y., Sano, Y., Kaneko, T., Abe, I., Noguchi,
H., Ito, K., 2004. Enzymatic reactions by five chalcone synthase homologs
from hop (Humulus lupulus L.). Bioscience, Biotechnology and
Biochemistry 68, 1142-1145.
The
enzyme activities encoded in five cDNAs for chalcone synthase (CHS)
homologs from hop were investigated. Only valerophenone synthase (VPS)
and CHS_H1 showed both naringenin-chalcone and phlorisovalerophenone
forming activity. Naringenin-chalcone production by VPS was much lower
than by CHS_H1. Therefore, it is highly possible that flavonoid depends
mainly on CHS_H1, while bitter acid biosynthesis depends mainly on VPS
and CHS_H1.
Zurück zum Text
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Okada, Y., Yamazaki, Y., Suh, D.-Y., Sankawa, U.,
Ito, K., 2001b. Bifunctional activities of valerophenone synthase in hop (Humulus
lupulus L.). Journal of the American Society of Brewing Chemists 59,
163-166.
Valerophenone synthase (VPS) catalyzes the
biosynthesis of phlorisovalerophenone, which is a precursor of hop (Humulus
lupulus L.) resin. The VPS gene is specifically expressed in the
lupulin gland, and the enzyme has a high similarity to chalcone synthase
(CHS). CHS catalyzes the formation of naringenin-chalcone; it is
considered a precursor of the prenylflavonoids, which accumulate in the
lupulin gland. We tried to confirm whether VPS has CHS activity using
the purified translation product of the VPS gene expressed in
Escherichia coli. The results indicated that VPS had CHS activity
and that the enzyme activity of VPS was about six times stronger than
the CHS activity. Therefore, VPS seems to contribute to the biosynthesis
of both resin and the prenylflavonoids in the lupulin gland, and the
difference in the bifunctional activities of VPS might have some effect
on the relative amounts of resin and prenylflavonoid in the gland.
Furthermore, we investigated (by Southern analysis) the existence of CHS
genes, which contribute to the polyphenol biosynthesis in the bract and
bracteole. We confirmed that there are two types of VPS genes, and also
more than two other types of genes, that have high similarity to the VPS
gene and might be the CHS gene.
Zurück zum Text
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Paniego, N. B., Zuurbier, K. W. M., Fung, S.-Y.,
Van der Heijden, R., Scheffer, J. J. C., Verpoorte, R., 1999.
Phlorisovalerophenone synthase, a novel polyketide synthase from hop (Humulus
lupulus L.) cones. European Journal of Biochemistry 262, 612-616.
Phlorisovalerophenone synthase (VPS), a novel aromatic polyketide
synthase, was purified to homogeneity from 4.2 mg protein extract
obtained from hop (Humulus lupulus L.) cone glandular hairs. The
enzyme uses isovaleryl-CoA or isobutyryl-CoA and three molecules of
malonyl-CoA to form phlorisovalerophenone or phlorisobutyrophenone,
intermediates in the biosynthesis of the hop bitter acids (alpha- and
beta-acids). VPS is an homodimeric enzyme, with subunits of 45 kDa. The
pI of the enzyme was 6.1. Km values of 4 µM for
isovaleryl-CoA, 10 µM for isobutyryl-CoA and 33 µM for malonyl-CoA, were
found. The amino- acid sequences of two peptides, obtained by digestion
of VPS, showed that the enzyme is highly homologous to plant chalcone
synthases. The functional and structural relationship between VPS and
other aromatic polyketide synthases is discussed.
Zurück zum Text
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Raharjo, T. J., Chang, W. T., Verberne, M. C.,
Peltenburg-Looman, A. M. G., Linthorst, H. J. M., Verpoorte, R., 2004.
Cloning and over-expression of a cDNA encoding a polyketide synthase from
Cannabis sativa. Plant Physiology and Biochemistry 42, 291-297.
A polyketide
synthase has been suggested to play an important role in cannabinoid
biosynthesis in Cannabis sativa L. This enzyme catalyzes the
biosynthesis of olivetolic acid, one of the precursors for cannabinoid
biosynthesis. Using a reverse transcriptase-polymerase chain reaction
(RT-PCR) based on the DNA homology of chalcone synthase (EC 2.3.1.156)
and valerophenone synthase (EC 2.3.1.156) of hop (Humulus lupulus),
a cDNA encoding a polyketide synthase in C. sativa was
identified. The coding region of the gene is 1170 bp long encoding a 389
amino acid protein of a predicted 42.7 kDa molecular mass and with a pI
of 6.04. The gene shares a high homology with a chalcone synthase gene
of H. lupulus, 85% and 94% homology on the level of DNA and
protein, respectively. Over-expression of the construct in
Escherichia coli M15 resulted in a 45 kDa protein. The protein has
chalcone synthase activity as well as valerophenone synthase activity, a
chalcone synthase-like activity. Using n-hexanoyl-CoA and
malonyl-CoA as substrates did not give olivetol or olivetolic acid as a
product.
Zurück zum Text
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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 zum Text
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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.
Zurück zum Text
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Zuurbier, K. W. M., Fung, S.-Y., Scheffer, J. J.
C., Verpoorte, R., 1995.
Formation of aromatic
intermediates in the biosynthesis of bitter acids in Humulus lupulus.
Phytochemistry 38, 77-82.
A new
hypothesis concerning the formation of aromatic intermediates in the
biosynthesis of hop bitter acids was tested. Two phloroglucinol
derivatives, 2-(3-methylbutanoyl)-1,3,5-benzenetriol
(phlorisovalerophenone = PIVP) and 2-(2-
methylpropanoyl)-1,3,5-benzenetriol (phlorisobutyrophenone = PIBP), were
detected in hop extracts. The formation of these compounds was shown to
occur in vitro in hop protein extracts using the precursors
malonyl-CoA plus isovaleryl-CoA or isobutyryl-CoA, respectively. We
conclude that PIVP and PIBP are probably the first aromatic
intermediates formed in the biosynthesis of humulone/lupulone and
cohumulone/colupulone, respectively. We suggest that the reactions are
catalyzed by a chalcone synthase-like enzyme. The enzyme chalcone
synthase and its activity were found to be present in hop protein
extracts. The formation of naringenin, PIVP and PIBP in various hop
protein extracts obtained during the development from flower bud to ripe
cone was compared.
Zurück zum Text
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Zuurbier, K. W. M., Leser, J., Berger, T.,
Hofte, A. J. P., Schröder, G., Verpoorte, R., Schröder, J., 1998.
4-Hydroxy-2-pyrone
formation by chalcone and stilbene synthase with nonphysiological
substrates.
Phytochemistry
49, 1945-1951.
Valerophenone synthase (VPS) is a polyketide synthase that
catalyzes the formation of the phloroglucinol derivatives in the synthesis
of the bitter acids in hop (Humulus lupulus). The reaction
uses isovaleryl-CoA or isobutyryl-CoA, but otherwise it is identical to that
of the chalcone synthase in flavonoid biosynthesis. Our study showed that
chalcone synthase can perform the function of VPS, but not perfectly,
because the majority of the reactions terminated after two condensation
reactions (products: 4-hydroxy-2-pyrone derivatives). The same experiments
with stilbene synthase yielded exclusively the 4-hydroxy-2-pyrone
derivatives, not the products expected from three condensation reactions.
The results are discussed in the context of the functional diversity and
evolution in the family of CHS-related polyketide synthases.
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CHS-Typ Ring-Faltung, aber andere Substrate
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