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(Last
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Biphenyl Synthase (BIS, EC 2.3.2.177) from Mountain Ash (Sorbus aucuparia)
(Beerhues et al., 2006;
Liu et
al., 2007)
Sorbus aucuparia
Biphenyls (e.g. aucuparin) and dibenzofurans (e.g. malusfuran) are phytoalexins of economically important
Rosaceae, in particular in the subfamily Maloideae (Kokubun
and Harborne, 1994; Kokubun and
Harborne, 1995; Kokubun et al., 1995); they
contribute to defense reactions against pathogen attack. Although
there is a report that the two substance groups do not occur simultaneously (Kokubun and
Harborne, 1995), experiments with tissue cultures from Malus x domestica
(apple) showed the simultaneous induction of both, and the similar substitution
patterns (see below) suggested that biphenyl-type compounds are precursors of
the dibenzofurans (Borejsza-Wysocki
et al., 1999), e.g. the malusfuran shown in the figure below. Precursor feeding studies (reviewed in
Sultanbawa, 1980)
suggested a polyketide synthase type biosynthetic reaction, leading to a
proposal that the biosynthesis involved a benzoyl-CoA starter and three
condensation reactions with malonyl-CoA, followed by a stilbene synthase-type
ring-folding (Schröder, 2000).
Sorbus aucuparia
(mountain ash, Eberesche, Vogelbeere,
Tree of the year in 1997 in Germany!) and its cell culture are interesting systems to study the
polyketide synthase (PKS) postulated in the biosynthesis. Wikipedia contains a fairly
good page in the German version (Vogelbeere),
but the English page (Sorbus aucuparia)
is not that good. However, even the German page does not mention the
biphenyl phytoalexins that are the focus of our interest. It also does not
mention parasorboside, the bitter tasting compound in Sorbus aucuparia
berries. The backbone of this natural product is also synthesized via a type III
PKS, but from acetyl-CoA and with only two condensation reactions:
More....
The biphenyl synthase (BIS)
enzyme reaction was demonstrated in extracts from cell cultures induced by
treatment with yeast extract (Liu et al.,
2004), and the same group recently succeeded in cloning the cDNA (Liu
et al., 2007). The enzyme shares about 60% identity with other members of
the PKSIII superfamily, and a
phylogenetic analysis indicates that it is most closely related to
benzophenone synthase (BPS), an enzyme that also uses benzoyl-CoA and carries
out three condensations, but the ring-folding is of the chalcone synthase (CHS)
type: more....
It is noteworthy that the BIS has no activity with 4-coumaroyl-CoA, and thus cannot
synthesize resveratrol, the product of a stilbene synthase-type reaction with that
substrate.
An interesting question is whether the biphenyl synthase (BIS)
is likely to use the
aldol switch mechanism discovered with the
stilbene synthase (STS) from
Scots pine (Pinus sylvestris) (Austin
et al., 2004). However, the amino acid residues
characteristic for the aldol switch appear to be missing in BIS, and thus it may
be possible that there may be alternative mechanismen for the STS-type
ring-folding (more...).
Update
06.October 2009:
A recent publication described
the cloning of two more BIS cDNAs, and a more detailed characterization of the
reactions of these proteins (BIS2 and BIS3) and BIS1 with 2-hydroxybenzoyl-CoA:
the synthesis of 4-hydroxycoumarin by this type III PKS:
more...
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References
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Liu, B., Raeth, T., Beuerle,
T., Beerhues, L., 2007. Biphenyl synthase, a novel type III polyketide
synthase. Planta 225, 1495-1503.
Biphenyls and dibenzofurans are the phytoalexins of the Maloideae, a
subfamily of the economically important Rosaceae. The carbon skeleton of
the two classes of antimicrobial secondary metabolites is formed by
biphenyl synthase (BIS). A cDNA encoding this key enzyme was cloned from
yeast-extract-treated cell cultures of Sorbus aucuparia. BIS is a
novel type III polyketide synthase (PKS) that shares about 60% amino
acid sequence identity with other members of the enzyme superfamily. Its
preferred starter substrate is benzoyl-CoA that undergoes iterative
condensation with three molecules of malonyl-CoA to give
3,5-dihydroxybiphenyl via intramolecular aldol condensation. BIS did not
accept CoA-linked cinnamic acids such as 4-coumaroyl-CoA. This substrate,
however, was the preferential starter molecule for chalcone synthase (CHS)
that was also cloned from S. aucuparia cell cultures. While BIS
expression was rapidly, strongly and transiently induced by yeast
extract treatment, CHS expression was not. In a phylogenetic tree, BIS
grouped together closely with benzophenone synthase (BPS) that also uses
benzoyl-CoA as starter molecule but cyclizes the common intermediate via
intramolecular Claisen condensation. The molecular characterization of
BIS thus contributes to the understanding of the functional diversity
and evolution of type III PKSs.
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Liu, B., Beuerle, T., Klundt,
T., Beerhues, L., 2004. Biphenyl synthase from yeast-extract-treated cell
cultures of Sorbus aucuparia. Planta 218, 492-496.
Biphenyls and dibenzofurans are the phytoalexins of the Maloideae, a
subfamily of the economically important Rosaceae. The biphenyl aucuparin
accumulated in Sorbus aucuparia L. cell cultures in response to
yeast extract treatment. Incubation of cell-free extracts from
challenged cell cultures with benzoyl-CoA and malonyl-CoA led to the
formation of 3,5-dihydroxybiphenyl. This reaction was catalysed by a
novel polyketide synthase, which will be named biphenyl synthase. The
most efficient starter substrate for the enzyme was benzoyl-CoA.
Relatively high activity was also observed with 2-hydroxybenzoyl-CoA but,
instead of the corresponding biphenyl, the derailment product
2-hydroxybenzoyltriacetic acid lactone was formed.
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Beerhues, L., Liu, B.,
Raeth, T., Klundt, T., Beuerle, T., Bocola, M., 2006.
Benzoic acid-specific type III polyketide synthases. In: Rimando, A. M.,
Baerson, S. R. (Eds.), Polyketides: Biosynthesis, Biological Activities and
Genetic Engineering, American Chemical Society, Washington, D.C., pp.
97-108.
Benzophenone
synthase (BPS) and biphenyl synthase (BIS) catalyze the formation of
the same linear tetraketide from benzoyl-CoA and three molecules of
malonyl-CoA. However, BPS cyclizes this intermediate via intramolecular
C6-C1 Claisen condensation, whereas BIS uses intramolecular C2-C7 aldol
condensation. Benzophenone derivatives include polyprenylated polycyclic
compounds with high pharmaceutical potential. Biphenyl derivatives are
the phytoalexins of the economically important Maloideae.
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Austin, M. B., Bowman, M.
E., Ferrer, J.-L., Schröder, J., Noel, J. P., 2004. An aldol switch
discovered in stilbene synthases mediates cyclization specificity of type
III polyketide synthases. Chemistry & Biology 11, 1179-1194.
Stilbene synthase (STS) and chalcone synthase (CHS) each catalyze the
formation of a tetraketide intermediate from a CoA-tethered phenylpropanoid
starter and three molecules of malonyl-CoA, but use different cyclization
mechanisms to produce distinct chemical scaffolds for a variety of plant
natural products. Here we present the first STS crystal structure, and
identify, by mutagenic conversion of alfalfa CHS into a functional stilbene
synthase, the structural basis for the evolution of STS cyclization
specificity in type III polyketide synthase (PKS) enzymes. Additional
mutagenesis and enzymatic characterization confirms that electronic effects
rather than steric factors balance competing cyclization specificities in CHS
and STS. Finally, we discuss the problematic in vitro reconstitution of
plant stilbenecarboxylate pathways, using insights from existing biomimetic
polyketide cyclization studies to generate a novel mechanistic hypothesis to
explain stilbenecarboxylate biosynthesis.
Reprint request
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Borejsza-Wysocki,
W., Lester, C., Attygalle, A. B., Hrazdina, G., 1999. Elicited cell
suspension cultures of apple (Malus x domestica) cv. Liberty produce
biphenyl phytoalexins. Phytochemistry 50, 231-235.
Yeast extract treated cell suspension cultures from a scab (Venturia
inaequalis) resistant apple cultivar, Malus x domestica
cv. Liberty produce dibenzofuran and biphenyl compounds as part of
their defense system against fungal invasion. We have isolated and
identified three biphenyl derivatives, 4-hydroxy-3,5- dimethoxybiphenyl
(aucuparin), 2',4,-dihydroxy-3,5- dimethoxybiphenyl
(2'-hydroxy-aucuparin) and 2'-O-beta-D-
glucopyranosyl-4-hydroxy-3,5-methoxybiphenyl (2'-o-beta-D-
glucopyranosylaucuparin) from the cells and the medium and show here
their chemical properties. Although this is the first identification of
2'-glucopyranosylaucuparin, its aglycone, 2'- hydroxyaucuparin and
aucuparin have been reported previously [Kokubun, T., Harborne, J.B.,
Phytochemistry, 1995, 40, 1649- 1654.] from fungus infected wood of
Malus species. Production of an array of dibenzofuran and biphenyl
derivatives in response to fungal attack may he an important part of the
disease resistance mechanism of scab resistant apple cultivars.
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Kokubun, T., Harborne, J.
B., 1994. A survey of phytoalexin induction in leaves of the Rosaceae by
copper ions. Zeitschrift für Naturforschung 49c, 628-634.
The
leaves of 130 species of Rosaceae were surveyed for phytoalexin
induction. Both biotic and abiotic induction was examined and antifungal
compounds were detected in 47 species. However, these compounds appeared
to be constitutive metabolites, released from bound phenolic materials
already present in the leaf. In Pyrus, hydroquinone was produced from
the hydrolysis of arbutin present in the vacuole before inoculation. In
most other species, the fungitoxic agents were mainly catechin-like
derivatives, apparently released from the tannins present within the
leaf. By contrast, the synthesis in the leaf of the characteristic
biphenyl or benzofuran phytoalexins which are produced in sapwood, was
confined to a very few species. The biphenyl aucuparin was identified as
a phytoalexin from the leaves of Sorbus aucuparia.
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Kokubun, T., Harborne, J.
B., 1995. Phytoalexin induction in the sapwood of plants of the Maloideae
(Rosaceae): biphenyls or dibenzofurans. Phytochemistry 40, 1649-1654.
Following fungal inoculation or natural infection, five biphenyl
phytoalexins (aucuparin and its 2' and 4' oxygenated derivatives) were
induced variously in the sapwood of Aronia, Chaenomeles, Eriobotrya,
Malus (three spp.) and of Sorbus aucuparia. By contrast, 14 dibenzofuran
phytoalexins were induced variously in sapwood of Cotoneaster (7 spp.),
Crateagus, Cydonia, Mespilus, Photinia, Pseudocydonia, Pyracantha, Pyrus
and two Sorbus spp. (S. chamaemespilum and S. domestica). These were
five cotonefurans, three eriobofurans, five pyrufurans and a 2,3,4,7,8-
pentaoxygenated dibenzofuran trimethyl ether. No plant has yet been
found to produce both types of phytoalexin, although o-hydroxybiphenyls
are theoretically precursors of the dibenzofurans. The ability to
synthesize either biphenyls or dibenzofurans appears to be
genus-specific, except in the case of Sorbus. In 18 of the 38 species
tested, these phytoalexins were accompanied by constitutive antifungal
phenolics, most of which appeared to be released from bound (glycosidic)
forms during the infection process. These were identified variously as
hydroquinone, p-hydroxyacetophenone, acetovanillone,
5,7-dihydroxychromone, chrysin, sakuranetin and naringenin. Woody
members of the subfamilies Prunoideae and Spiraeoideae failed to yield
any phytoalexins on induction, but did contain constitutive antifungal
compounds. The limited frequency of the phytoalexin response within the
family as a whole is considered in relation to the accumulation of
constitutive antifungal agents in these plants.
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Kokubun, T., Harborne, J.
B., Eagles, J., Waterman, P. G., 1995. Antifungal biphenyl compounds are
the phytoalexins of the sapwood of Sorbus aucuparia.
Phytochemistry 40, 57-59.
An
examination of the sapwood tissue of Sorbus aucuparia L. has
revealed that aucuparin and its derivatives are essentially
absent from healthy tissue, and are only produced as phytoalexins
following fungal infection. Five biphenyls were identified: aucuparin,
2'-methoxyaucuparin, 4'-methoxyaucuparin, 2'- hydroxyaucuparin and
isoaucuparin (2'-hydroxy-3,5- dimethoxybiphenyl). The latter is a new
phytoalexin. A survey of 11 individual Sorbus trees showed that not all
these compounds are necessarily produced in the phytoalexin response.
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Schröder, J., 2000. The
family of chalcone synthase-related proteins: functional diversity and
evolution. Recent Advances in Phytochemistry 34, 55-89.
CONCLUSIONS: Results in the last few years showed that the
well-known chalcone synthase (CHS) is only one example from a family of
plant polyketide synthases. Other members of the family which are
identified by function and sequences are the stilbene synthases (STS),
acridone synthase (ACS), and a pyrone synthase (2PS); all of these
proteins share about 65-70% identity with CHS. The properties of several
other enzymes suggest that they are members of the protein family, and
precursor feeding studies suggest that the number may be much larger
than suspected so far. The diversity of functions is based on different
substrate specificities, variations in the number of condensation
reactions, folding of intermediates to different products, and
modification of intermediates by other enzymes.
The recently published first crystal structure of a CHS raises hopes
that it will be possible to understand at the protein sequence level the
programming of the proteins for the various functions; this then will
facilitate the design of enzymes synthesizing new products.
The understanding of the evolution of the protein family is still
rudimentary. The available data suggest that the functional diversity
known in present-day plants could be the results of fairly recent
developments from CHS by gene duplication and mutation. The presence of
CHS-related sequences in bacteria indicates that the basic function unit
predated the evolution of plants. The recent functional identification
of such a protein from Streptomyces griseus suggests that
the functional diversity in bacteria may even be larger than in plants.
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Sultanbawa, M. U. S.,
1980. Xanthonoids of tropical plants.
Tetrahedron 36,
1465-1506.
No Abstract available.
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