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(Last
modification: 23. October 2010)
Sorgoleone Biosynthesis (Sorghum bicolor)
Alkylresorcinol Synthases (ARS)
(Cook et al., 2006,
2010)
New October
23, 2010: have a look at a new publication providing additional information:
Baerson et al., 2010.
A look at the postulated polyketide synthase (PKS)
reaction indicates that this should be a stilbene synthase (STS) type reaction
(i.e. aldol condensation) with a
long chain fatty acid CoA-ester as starter substrate, as shown below. This was
also proposed in a review discussing the potential catalytic diversity of plant
type III PKS (Austin and Noel, 2003).
Fig. 1
Biosynthesis of Sorgoleone:
role of a polyketide synthase
The work by Cook et al. (2006)
confirmed this
prediction: A root-hair specific EST collection (EST = expressed
sequence tag) was established, and sequences
for three type III PKS proteins were identified.
A publication
describing the molecular and functional characterization, and much more, is now
out (March 2010), and the pages linked below summarize some of the important
findings:
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This page:
Alkylresorcinol synthases (ARS) in sorgoleone biosynthesis:
more...
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Type III PKS transcripts
preferentially expressed in root hairs:
more...
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Activities and substrate
preferences of ARS type III PKS from Sorghum bicolor:
more...
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Molecular modeling of
Sorghum ARS:
more...
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ARS from rice (Oryza
sativa): functional comparison with Sorghum ARS:
more...
- Update July 2010: ARS or ARAS ?
more...
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ARS from Sorghum and rice (Oryza sativa),
identification of functionally important amino acids:
more...
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RNAi mediated repression of ARS1 and ARS2 in Sorghum bicolor:
more...
Another interesting point emerged from the
studies with Sorghum, and can be mentioned here already: the sequences showed a high degree of similarity to a
small family of putative PKSs from rice. Their functions are not yet known, but
it seems very well possible that they have similar enzyme functions: rice does contain alkylresorcinol compounds related to the basic structure in
sorgoleone (Suzuki et al., 1996;
1998), and
precursor feeding studies suggested that a similar PKS reaction as in Sorghum
should be a key enzyme in their biosynthesis (Suzuki
et al., 2003). The publication by Cook et al. (2010) actually describes the
functional identification of three rice type III PKS: they are indeed
alkylresorcinol synthases:
more....
It
seems very likely that similar activities are present in
other monocotyledons, because alkylresorcinols have been reported from
barley (Garcia
et al., 1997; Zarnowski et al., 2002;
Zarnowski and Suzuki, 2004),
wheat (Chen
et al., 2004; Landberg et al., 2006), and
rye (Suzuki
et al., 1999; Deszcz and Kozubek, 2000;
Ross et al., 2001;
Chen et al., 2004).
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Direct links to other enzymes carrying out aldol condensations
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Other type III PKS
with substrate preferences for long-chain
CoA-esters
in plants, bacteria, and fungi
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Alkylresorcinols
and
long-chain pyrones in the bacterium Azotobacter vinelandii:
more...
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Alkylresorcinol
Streptomyces
griseus: more...
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Pyrone synthases in the bacteria
Mycobacterium tuberculosis and
Bacillus subtilis: more...
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CsyA: Pyrone synthases in the fungus
Aspergillus oryzae: more...
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References
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Baerson, S. R., Schröder, J., Cook, D.,
Rimando, A. M., Pan, Z., Dayan, F. E., Noonan, B. P., Duke, S. O.,
2010. Alkylresorcinol
biosynthesis in plants: new insights from an ancient enzyme family?
Plant Signaling and Behavior 5, Oct 18 [Epub ahead of print]
Alkylresorcinols are members of an extensive family of bioactive
compounds referred to as phenolic lipids, which occur primarily in
plants, fungi and bacteria. In plants, alkylresorcinols and their
derivatives are thought to serve important roles as phytoanticipins and
allelochemicals, although direct evidence for this is still somewhat
lacking. Specialized type III polyketide synthases (referred to as
'alkylresorcinol synthases'), which catalyze the formation of
5-alkylresorcinols using fatty acyl-CoA starter units and malonyl-CoA
extender units, have been characterized from several microbial species,
however until very recently little has been known concerning their plant
counterparts. Through the use of sorghum and rice EST and genomic data
sets, significant inroads have now been made in this regard. Here we
provide additional information concerning our recent report on the
identification and characterization of alkylresorcinol synthases from
Sorghum bicolor and Oryza sativa, as well as a brief
consideration of the emergence of this intriguing subfamily of enzymes.
Reprint request
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Cook, D., Rimando, A. M., Clemente, T. E.,
Schröder, J., Dayan, F. E., Nanayakkara, N. P. D., Pan, Z., Noonan, B.
P., Fishbein, M., Abe, I., Duke, S. O., Baerson, S. R., 2010: Alkylresorcinol
synthases from Sorghum bicolor involved in the biosynthesis of
the allelopathic benzoquinone sorgoleone.
Plant Cell 22, 867-878.
Sorghum bicolor
is considered to be an allelopathic crop species, producing phytotoxins
such as the lipid benzoquinone sorgoleone, which likely accounts for
many of the allelopathic properties of Sorghum spp. Current
evidence suggests that sorgoleone biosynthesis occurs exclusively in
root hair cells and involves the production of an alkylresorcinolic
intermediate (5-[(Z,Z)-8',11',14'-pentadecatrienyl]resorcinol) derived
from an unusual 16:3{Delta}9,12,15 fatty acyl-CoA starter unit. This led
to the suggestion of the involvement of one or more alkylresorcinol
synthases (ARSs), type III polyketide synthases (PKSs) that produce
5-alkylresorcinols using medium to long-chain fatty acyl-CoA starter
units via iterative condensations with malonyl-CoA. In an effort to
characterize the enzymes responsible for the biosynthesis of the
pentadecyl resorcinol intermediate, a previously described expressed
sequence tag database prepared from isolated S. bicolor (genotype
BTx623) root hairs was first mined for all PKS-like sequences.
Quantitative real-time RT-PCR analyses revealed that three of these
sequences were preferentially expressed in root hairs, two of which (designated
ARS1 and ARS2) were found to encode ARS enzymes capable of accepting a
variety of fatty acyl-CoA starter units in recombinant enzyme studies.
Furthermore, RNA interference experiments directed against ARS1 and ARS2
resulted in the generation of multiple independent transformant events
exhibiting dramatically reduced sorgoleone levels. Thus, both ARS1 and
ARS2 are likely to participate in the biosynthesis of sorgoleone in
planta. The sequences of ARS1 and ARS2 were also used to identify
several rice (Oryza sativa) genes encoding ARSs, which are likely
involved in the production of defense-related alkylresorcinols.
Link to the sequences of the
alkylresorcinol synthases
Reprint request
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Cook, D., Dayan, F. E., Rimando, A. M.,
Nanayakkara, N. P. D., Pan, Z., Duke, S. O., Baerson, S. R., 2006.
Molecular and biochemical characterization of novel polyketide synthases
likely to be involved in the biosynthesis of sorgoleone. In: Rimando, A.
M., Baerson, S. R. (Eds.), Polyketides: Biosynthesis, Biological Activities
and Genetic Engineering, American Chemical Society, Washington, D.C., pp.
141-151.
Sorgoleone, an oily exudate
secreted from the root hairs of sorghum (Sorghum bicolor (L.)
Moench), acts as a potent allelochemical. Its phytotoxic properties make
the elucidation of the biosynthetic enzymes participating in this
pathway desirable. Previous studies suggest that the biosynthetic
pathway of sorgoleone involves a polyketide synthase as well as a fatty
acid desaturase, an O-methyl transferase, and a cytochrome P450
monooxygenase. This polyketide synthase is proposed to use a novel long
chain fatty acyl-CoA (C16:3) as a starter unit and catalyzes three
iterative condensation reactions using malonyl-CoA to form a transient
linear tetraketide that cyclizes to form a pentadecatriene resorcinol.
To identify the polyketide synthase gene(s) involved in the biosynthesis
of these alkylresorcinols, a root-hair specific EST (expressed sequence
tag) collection was mined for potential candidates. A total of nine
polyketide synthase-like ESTs were identified representing five unique
contigs, three of which were preferentially expressed in root hairs. The
molecular and biochemical characterization of these three candidate
polyketide synthases are presented, two of which represent a novel type
of type III plant polyketide synthase.
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Austin, M. B., Noel, J. P., 2003.
The chalcone synthase superfamily of type III polyketide synthases. Natural
Product Reports 20, 79-110.
Covering 19702001. This
review covers the functionally diverse type III polyketide synthase (PKS)
superfamily of plant and bacterial biosynthetic enzymes, from the
discovery of chalcone synthase (CHS) in the 1970s through the end of
2001. A broader perspective is achieved by a comparison of these
CHS-like enzymes to mechanistically and evolutionarily related families
of enzymes, including the type I and type II PKSs, as well as the
thiolases and -ketoacyl synthases of fatty acid metabolism. As CHS is
both the most frequently occurring and best studied type III PKS, this
enzyme's structure and mechanism is examined in detail. The in vivo
functions and biological activities of several classes of plant natural
products derived from chalcones are also discussed. Evolutionary
mechanisms of type III PKS divergence are considered, as are the
biological functions and activities of each of the known and
functionally divergent type III PKS enzyme families (currently twelve in
plants and three in bacteria). A major focus of this review is the
integration of information from genetic and biochemical studies with the
unique insights gained from protein X-ray crystallography and homology
modeling. This structural approach has generated a number of new
predictions regarding both the importance and mechanistic role of
various amino acid substitutions observed among functionally diverse
type III PKS enzymes.
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Chen, Y., Ross, A. B., Aman, P., Kamal-Eldin,
A., 2004. Alkylresorcinols as markers of whole grain wheat and rye in
cereal products. Journal of Agricultural and Food Chemistry 52, 8242-8246.
The alkylresorcinol
(AR) content of Swedish wheat grain samples, as well as of cereal
ingredients and cereal foods containing wheat and rye, was determined.
The average total AR content in Swedish wheat was 412 µg/g (ranging
between 227 and 639 µg/g), which is lower than that in Swedish rye
analyzed in a previous study. The relative composition of AR homologues
was consistent for wheat samples and differed markedly from that of rye.
Notably, the ratio of the homologues C17:0/C21:0 was <0.1 in wheat and
>1.0 in rye, indicating that it can be used to distinguish between those
two cereals. The AR content in cereal foods commonly consumed in Sweden
varied widely, from nondetectable levels in white wheat flour and
products not containing the outer parts of wheat and/or rye to >900 µg/g
in some whole grain rye products. AR content in cereal foods was
calculated from their recipes using average AR values for the cereal
ingredients determined in this study. As there was a good correlation
between calculated and analyzed AR levels in cereal foods (R 2 = 0.91),
it is possible to estimate the proportion of whole grain wheat and/or
rye in a given cereal product on the basis of AR content and C17:0/C21:0
ratio. ARs appear to be good markers of whole grain wheat and rye in
foods, and their analysis may be an objective way to identify foods rich
in whole grain wheat and/or rye or brans thereof.
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Magnucka, E. G., Suzuki, Y., Pietr, S. J., Kozubek, A., Zarnowski,
R., 2007. Effect of norflurazon on resorcinolic lipid metabolism in rye
seedlings. Zeitschrift für Naturforschung 62c, 239-245.
Norflurazon is a selective pyridazinone herbicide excessively employed
in the control of many annual grasses and broad-leaved weeds. This
chemical causes plant bleaching due to the inhibition of the carotenoid
pigment biogenesis as well as induces irreparable changes to
chloroplasts, which are considered the organelles where the biosynthesis
of resorcinolic lipids takes place. Resorcinolic lipids, a group of
phenolic compounds, constitute not only an essential part of the plant
antifungal defense system, but also are an important component of the
human cereal diet. The aim of this study was to investigate the effect
of norflurazon on the biosynthesis of resorcinolic lipids in 5-day-old
rye plants (Secale cereale L.) that were grown at three different
temperatures under light or dark conditions. At all tested temperatures,
norflurazon decreased the fresh biomass of light-grown rye seedlings and
increased the weight of plants grown in darkness. Compared with
respective controls, this herbicide caused an increase in total content
of alkylresorcinols in both green and etiolated plants with the
exception of dark-grown norflurazon-treated rye at 29 degrees C. The
general level of saturated homologues was markedly decreased by
norflurazon in all etiolated plants and in light-grown seedlings at 15
degrees C. Independent of thermal and light conditions, in all
norflurazon-treated samples two alkylresorcinol derivatives predominated:
1,3-dihydroxy-5-n-heptadecylbenzene and
1,3-dihydroxy-5-n-nonadecylbenzene. Thus, our results suggest that
norflurazon affected the metabolism of alkylresorcinols in rye seedlings
and its action was dependent on external stimuli.
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Dayan, F. E., Kagan, I. A., Rimando, A. M.,
2003. Elucidation of the biosynthetic pathway of the allelochemical
sorgoleone using retrobiosynthetic NMR analysis. Journal of Biological
Chemistry 278, 28607-28611.
NMR analyses of the labeling
pattern obtained using various 13C-labeled precursors indicated that
both the lipid tail and the quinone head of sorgoleone, the main
allelopathic component of the oily root exudate of Sorghum bicolor,
were derived from acetate units, but that the two moieties were
synthesized in different subcellular compartments. The 16:3 fatty acid
precursor of the tail is synthesized by the combined action of
fatty-acid synthase and desaturases most likely in the plastids. It is
then exported out of the plastids and converted to 5-pentadecatriene
resorcinol by a polyketide synthase. This resorcinol intermediate was
identified in root hair extracts. The lipid resorcinol intermediate is
then methylated by a S-adenosylmethionine-dependent O-methyltransferase
and subsequently dihydroxylated by a P450 monooxygenase to yield the
reduced form of sorgoleone.
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Deszcz, L., Kozubek, A., 2000. Higher
cardol homologs (5-alkylresorcinols) in rye seedlings. Biochimica et
Biophysica Acta 1483, 241-250.
The occurrence of
alkylresorcinols, polyketide compounds that in the same homologous
series as cardol isolated from Anacardium occidentale (cashew) or
bilobol from Ginkgo biloba which are derivatives of
1,3-dihydroxy-5-alk(en)ylbenzene, have been demonstrated in developing
rye (Secale cereale L.) kernels. The 3-day-old seedlings grown in
sterile conditions already contain detectable amounts of phenolic
compounds that were identified as alkylresorcinols. This fraction is the
mixture of saturated and enoic homologs of various lengths of the
aliphatic side chain. The composition of homologs is similar to that
determined in mature grains. The relatively high level of
alkylresorcinols in mitochondria and plastids (enhanced approximately
twice in the absence of light) suggests that their synthetic pathway
and/or biological function may be related to these cellular compartments.
Resorcinolic lipids, when present in the external medium, are taken up
by seedlings in the energy-dependent manner.
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Garcia, S., Garcia, C., Heinzen, H., Moyna,
P., 1997. Chemical basis of the resistance of barley seeds to pathogenic
fungi. Phytochemistry 44, 415-418.
The 5-(n)-alkylresorcinol
fraction of the epicuticular waxes of Hordeum vulgare seeds
appeared to be responsible for their in-born resistance to pathogenic
fungi such as Aspergillus niger and Penicillium crysogenum.
The antifungal properties of this fraction were evaluated qualitatively
and quantitatively with a novel bioassay where the extreme lipophilicity
of these compounds was taken into account. The minimum inhibitory
concentration in the fungi tested ranged from 5.6 to 10 micrograms cm-2
for the alkyresorcinols. The behaviour of the different cultivars
against these fungi could be predicted by measuring the natural amount
of resorcinols of each variety by TLC-scanning densitometry. The ranking
of cultivars thus established correlated well with the field behaviour
of each cultivar, providing a useful and rapid method for predicting the
behaviour against fungi of new varieties being developed.
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Landberg, R., Kamal-Eldin, A., Andersson,
R., Aman, P., 2006. Alkylresorcinol content and homologue composition in
durum wheat (Triticum durum) kernels and pasta products. Journal of
Agricultural and Food Chemistry 54, 3012-3014.
The total alkylresorcinol (AR)
content and relative homologue composition of 21 durum wheat (Triticum
durum) kernel samples, as well as 5 pasta products and the
corresponding flour mixtures, were determined. Durum wheat contained on
average 455 µg/g ARs, and the average relative homologue composition was
C17:0 (0.4%), C19:0 (14%), C21:0 (58%), C23:0 (21%), and C25:0 (6.5%).
The homologue composition was found to be relatively consistent among
samples, with durum wheat being different from common wheat by having a
higher proportion of the longer homologues. No differences in content or
homologue composition were observed in pasta products compared to flour
ingredients, showing that alkylresorcinols are stable during pasta
processing. The ratio of the homologues C17:0 to C21:0 was <0.02 for
whole grain durum wheat products, which is different from those of
common wheat (0.1) and rye (0.9).
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Ross, A. B., Kamal-Eldin, A., Jung, C.,
Shepherd, M. J., Aman, P., 2001. Gas chromatographic analysis of
alkylresorcinols in rye (Secale cereale L.) grains. Journal of the
Science of Food and Agriculture 81, 1405-1411.
A method was established for
fast and easy analysis of 5-n-alkylresorcinols in rye grains,
considering effects of milling (intact grains versus flour), extraction
solvent (ethyl acetates methanol and acetone), extraction volume per
gram of sample (20 and 40 ml) and extraction time (3, 6, 18 and 24 h).
For intact kernels, extraction of 1 g samples with ethyl acetate (40ml,
24h) is recommended. The ease of extraction is consistent with the
presence of alkylresorcinols in the outer layers of the kernel and their
absence in the endosperm. The extracts were analysed (without further
purification or derivatisation) by gas chromatography using methyl
behenate (C22:0, fatty acid methyl ester) as internal standard.
Alkylresorcinols were identified by gas chromatography/mass spectrometry,
and the major homologues were found to contain alkyl chains of C17:0,
C19:0, C21:0, C23:0 and C25:0. The methodology was then applied to
analyse the alkylresorcinol content in 15 rye cultivars grown at two
locations in Sweden. Total alkylresorcinol contents varied within the
range 549-1022 ?g g-1, and the average percentages of the different
alkylresorcinols were: 17:0, 23%; 19:0, 32%; 21:0, 26%; 23:0, 11%; and
25:0, 8%.
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Suzuki, Y., Esumi, Y., Hyakutake, H., Kono,
Y., Sakurai, A., 1996. Isolation of 5-(8'Z-heptadecenyl)-resorcinol from
etiolated rice seedlings as an antifungal agent. Phytochemistry 41,
1485-1489.
A new antifungal substance
against rice blast fungus (Pyricularia oryzae),
5-(8'Z-heptadecenyl)resorcinol, was isolated from etiolated rice
seedlings together with a mixture of its homologues with C13, C15, and
C17 saturated alkyl chains. Its structure was determined by 1NMR,
13C-NMR, and MS spectra. Its ED50 was ca 40 µg/ml. These
5-alkylresorcinols were newly produced after germination and the
concentration reached 50 µg g-1 fresh weight on day 6.
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Suzuki, Y., Esumi, Y., Saito, T.,
Kishimoto, Y., Morita, T., Koshino, H., Uzawa, J., Kono, Y., Yamaguchi, I.,
1998. Identification of 5-n-(2'-oxo)alkylresorcinols from etiolated rice
seedlings. Phytochemistry 47, 1247-1252.
Six
5-n-(2'-oxo)alkylresorcinols were isolated from etiolated rice seedlings.
The identification of these compounds was achieved by spectroscopy. From
a comparison of the double bond position between 5-n- alkyl- and
5-n-(2'-oxo)alkylresorcinols in rice seedlings, the predicted
biosynthetic pathway of 5-n-alkylresorcinols was supported.
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Suzuki, Y., Esumi, Y., Yamaguchi, I., 1999.
Structures of 5-alkylresorcinol-related analogues in rye. Phytochemistry
52, 281-289.
A series of
5-(2'-oxo)alkylresorcinols, 5-(2'- and 4'- hydroxy)alkylresorcinols, and
5,5'-(alkadiyl)diresorcinols were isolated from rye, each as a mixture
of homologues. The identification of these alkylresorcinol-related
analogues was achieved by analyses of the 1H-NMR and MS spectra. From
the identity of the analogues found, a possible biosynthetic pathway to
the 2'-oxoalkylresorcinols is proposed, based on a correlation of the
carbon-chain lengths between the alkylresorcinols and 2'-
oxoalkylresorcinols.
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Suzuki, Y., Kurano, M., Esumi, Y.,
Yamaguchi, I., Doi, Y., 2003. Biosynthesis of 5-alkylresorcinol in rice:
Incorporation of a putative fatty acid unit in the 5-alkylresorcinol carbon
chain. Bioorganic Chemistry 31, 437-452.
A previously unspecified "starter"
unit in the predicted biosynthesis pathway of 5-alkylresorcinols has now
identified as a fatty acid or its equivalent, using an efficient
5-alkylresorcinol production system of etiolated rice seedlings. Feeding
saturated, odd-carbon fatty acid ester substrates from C11 to C19
specifically and markedly increased the amount of the corresponding
5-alkylresorcinol homologs with even-carbon chains that are shorter by
one carbon than those of the supplied fatty acids. The amount of these
homologs depended on substrate concentration. Some of the homologs whose
amounts increased had linear carbon chains and the dodecyl homolog was
shown to be 5-n-dodecylresorcinol. Moreover, the 13C label in the
dodecyl homolog that was biosynthesized from the [1-13C]tridecanoate
substrate was localized on the C-5 carbon of the resorcinol ring. These
results obviously show that the fatty acid unit acts as a direct
precursor and forms the side-chain moiety of 5-n-alkylresorcinol via the
predicted biosynthesis pathway.
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Zarnowski, R., Suzuki, Y., 2004.
5-n-Alkylresorcinols from grains of winter barley (Hordeum vulgare
L.).
Zeitschrift für Naturforschung 59c, 315-317.
The resorcinolic lipid content and homologue
composition of winter barley grains harvested at two field locations
were evaluated. Depending on the crop location, the predominant
alkylresorcinols identified were 1,3-dihydroxy-5-n-heneicosylbenzene or
1,3-dihydroxy-5-n-pentacosylbenzene. Both resorcinol concentration and
their homologue profiles were diverse in samples harvested at different
fields indicating a prevailing role of the environment upon the
alkylresorcinol biosynthesis in cereals.
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Zarnowski, R., Suzuki, Y., Yamaguchi,
I., Pietr, S. J., 2002. Alkylresorcinols in barley (Hordeum vulgare
L. distichon) grains. Zeitschrift für Naturforschung 57c, 57-62.
This study was carried out to compare
grains of barley (Hordeum vulgare L. distichon) regarding
contents and compositions of 5-n-alkylresorcinols. Mixtures of
resorcinol homologues were isolated from acetone extracts from five
barley cultivars. These polyketide metabolites were identified by
chromatographic and spectroscopic means. The content and homologue
patterns among different varieties were similar. The predominant
compounds were 1,3-dihydroxy-5-n-heneicosylbenzene (C21:0),
1,3-dihydroxy-5-n-nonadecylbenzene (C19:0) and
1,3-dihydroxy-5-n-pentacosylbenzene (C25:0). The alkylresorcinol
concentrations, in contrast to their compositions, depended on
environmental and agricultural factors.
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