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(Last modification: 11.
June 2010)
Type III Polyketidsynthasen
(PKS) in Bakterien
Stilbensynthase (STS) und Pyron-Typ Ring-Faltung
Azotobacter vinelandii:
Biosynthese von Alkylresorcinolen und langkettigen Pyronen
(Funa
et al., 2006)
Die
Cysten von
Azotobacter vinelandii
(gehört zu einer Gruppe gram-negativer Stickstoff-Fixierer) enthält eine Reihe
von phenolischen Lipiden; eine Mischung aus Alkylresorcinolen und Alkylpyronen,
mit variablen aliphatischen Seitenketten (viele länger als 20 C-Atome) (Reusch
and Sadoff, 1979; Reusch and
Sadoff, 1983; Zarnowski
et al., 2004). Ähnliche Substanzen wurden auch in verschiedenen Pseudomonas
Stämmen identifiziert (Kozubek et
al., 1996).
Alkylresorcinole sind auch aus anderen Organismen bekannt
(Review:
Kozubek et al., 2001). Dazu
gehören auch einige interessante Beispiele in Pflanzen, und ihre Biosynthese
wird auf anderen Seiten diskutiert: Das allelophatische Sorgoleone in Sorghum
bicolor (mehr...) und
die Anacardsäuren und Urushiole in extrem allergenen Pflanzen (z.B. 'poison ivy'
usw., mehr...). Es ist
interessant, dass die verfügbare Evidenz klar dafür spricht, dass Typ III PKS
Schlüsselrollen in der Biosynthese spielen, in Bakterien und Pflanzen, und
vermutlich auch in Pilzen (mehr...)!
In Bakterien ist ein gut untersuchtes Beispiel aus
Azotobacter
vinelandii (Funa
et al., 2006). Diese Arbeit zeigte, dass die Bakterien wenigstens zwei Typ
III PKS enthalten, und dass sie verantwortlich sind für die Biosynthese der
Alkylresorcinole und Alkylpyrone, die in Cysten gefunden werden. ArsB und ArsC
sind eng verwandt: 71% identisch. Die prinzipiellen Ergebnisse sind hier zusammengefasst:
Fig.
1.
Overview: biosynthesis of long-chain alkylresorcinols and alkylpyrones in
Azotobacter vinelandii.
ArsA and ArsD (see also below) are the enzymes building the long-chain alkyls
used by the type III PKS ArsB and ArsC. Note that these two enzymes are not
identical in function: ArsB is capable of performing the cyclization to a
resorcinol, but ArsC is not. It would be nic to understand the mechanistic basis
for this difference, i.e. to identify the amino acids in the proteins which are
responsible.
Eine interessante Frage ist, ob dieses Enzym wohl den "aldol switch mechanism"
verwendet, der mit der Stilbensynthase (STS)
aus der Kiefer (Pinus sylvestris) entdeckt wurde:
Mehr...
Zum Seitenanfang
Organisation des
Ars Operon und Kopplung von Typ I FAS und Typ III PKS
(Miyanaga et al., 2008)
Dies sind
sehr interessante neuere Ergebnisse: Das Ars Operon enthält nicht nur
Gene für zwei Typ III PKS, sondern auch noch zwei Gene, die zusammen eine sehr
schöne Typ I PKS ausmachen. Die Organisation des Operons und seiner Komponenten
ist unten gezeigt. Der Trick des Systems, sehr ungewöhnlich:
die Typ I FAS synthetisiert die
langkettigen Fettsäuren, die dann direkt aus der ACP-gebundenen Form an das
aktive Cystein der Typ III PKS übergeben werden !!!
Fig. 2.
Organization of the ars operon in Azotobacter
vinelandii.
Zum
Seitenanfang
An addition, from
a publication in May 2009 (Segura
et al., 2009)
This work investigated the claim that the lack of
alkylresorcinols leads to "severely impaired cysts"
(Funa et al., 2006). They mutagenized (inactivated) arsA, and then looked for
cyst formation and desiccation tolerance. What did they find? The block of
alkylresorcinol formation by no means blocked cyst formation or desiccation
tolerance. And what is the explanation? It seems that Funa et al. did not
realize that the Azotobacter strain used by them was already a mutant impaired
in a function very important for cyst formation: it cannot synthesize alginates.
Quite interesting work, but you should look at the publication yourself. The
main conclusion, and that was rather convincing to me:
alkylresorcinols play a
structural role in the cysts, but they are not essential for cyst formation and
desiccation tolerance.
Andere Typ
III PKS mit Substrat-Präferenzen für langkettige CoA-Estern gibt es anscheinend häufig
in Pflanzen und Bakterien. Beispiele sind:
-
-
-
-
Alkylresorcinole und
langkettige Pyrone in dem Bakterium Azotobacter vinelandii:
Mehr...
-
Alkylresorcinol-
Streptomyces
griseus: Mehr...
-
Pyronsynthasen in dem Bakterium
Mycobacterium tuberculosis und
Bacillus subtilis: Mehr...
-
CsyA: Pyronsynthasen
in dem Pilz Aspergillus oryzae:
Mehr...
Links zu bakteriellen Typ III PKS
Zum Seitenanfang
Ausgewählte Zitate
-
Segura, D., Vite, O., Romero, Y., Moreno, S.,
Castaneda, M., Espin, G., 2009. Isolation and characterization of
Azotobacter vinelandii mutants impaired in alkylresorcinol synthesis:
alkylresorcinols are not essential for cyst desiccation resistance.
Journal of Bacteriology 191, 3142-3148.
During encystment of Azotobacter vinelandii, a family of
alkylresorcinols (ARs) and alkylpyrones (APs) are synthesized. In the
mature cyst, these lipids replace the membrane phospholipids and are
also components of the layers covering the cyst. In this study, A.
vinelandii strains unable to synthesize ARs were isolated after
mini-Tn5 mutagenesis. Cloning and nucleotide sequencing of the affected
loci revealed the presence of the transposons within the arsA gene of
the previously reported arsABCD gene cluster, which encodes a type I
fatty acid synthase. A mutant strain (SW-A) carrying an arsA mutation
allowing transcription of arsBCD was constructed and shown to be unable
to produce ARs, indicating that the ArsA protein is essential for the
synthesis of these phenolic lipids. Transcription of arsA was induced
200-fold in cells undergoing encystment, but only 14-fold in aged
cultures of A. vinelandii, in accordance with AR synthesis and
cyst formation percentages under the two conditions. Although it was
previously reported that the inactivation of arsB abolishes AR synthesis
and results in a failure in encystment, the arsA mutants were able to
form cysts resistant to desiccation. These data indicate that ARs play a
structural role in the exine layer of the cysts, but they are not
essential for either cyst formation or for desiccation resistance.
Zurück
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Funa, N., Ozawa, H., Hirata, A., Horinouchi, S.,
2006. Phenolic lipid synthesis by type III polyketide synthases is
essential for cyst formation in Azotobacter vinelandii. Proceedings
of the National Academy of Sciences of the United States of America 103,
6356-6361.
Cysts of
Azotobacter vinelandii are resting cells that are surrounded by a
protective coat, conferring resistance to various chemical and physical
agents. The major chemical components of the cyst coat are
alkylresorcinols, which are amphiphilic molecules possessing an aromatic
ring with a long aliphatic carbon chain. Although alkylresorcinols are
widely distributed in bacteria, fungi, plants, and animals, no enzyme
systems for their biosynthesis are known. We report here an ars operon in
A. vinelandii that is responsible for the biosynthesis of the
alkylresorcinols in the cysts. The ars operon consisted of four genes, two
of which encoded a type III polyketide synthase, ArsB and ArsC. In vitro
experiments revealed that ArsB and ArsC, sharing 71% amino acid sequence
identity, were an alkylresorcinol synthase and an alkylpyrone synthase,
respectively, indicating that ArsB and ArsC are not isozymes but
enzymatically distinct polyketide synthases. In addition, ArsB and ArsC
accepted several acyl-CoAs with various lengths of the side chain as a
starter substrate and gave corresponding alkylresorcinols and alkylpyrones,
respectively, which suggests that the mode of the ring folding is
uninfluenced by the structure of the starter substrates. The importance of
the alkylresorcinols for encystment was confirmed by gene inactivation
experiments; the lack of alkylresorcinols synthesis caused by ars mutations
resulted in the formation of severely impaired cysts, as observed by
electron microscopy.
Protein accession no.: ArsB =
YP_002800096 ;
ArsC =
YP_002800095
Zurück zum Text
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Miyanaga,
A., Funa, N., Awakawa, T., Horinouchi, S., 2008. Direct transfer of starter
substrates from type I fatty acid synthase to type III polyketide synthases
in phenolic lipid synthesis. Proceedings of the National Academy of
Sciences of the United States of America 105, 871-876.
Alkylresorcinols and alkylpyrones, which have a polar aromatic ring and a
hydrophobic alkyl chain, are phenolic lipids found in plants, fungi, and
bacteria. In the Gram-negative bacterium Azotobacter vinelandii,
phenolic lipids in the membrane of dormant cysts are essential for
encystment. The aromatic moieties of the phenolic lipids in A. vinelandii
are synthesized by two type III polyketide synthases (PKSs), ArsB and ArsC,
which are encoded by the ars operon. However, details of the synthesis of
hydrophobic acyl chains, which might serve as starter substrates for the
type III polyketide synthases (PKSs), were unknown. Here, we show that two
type I fatty acid synthases (FASs), ArsA and ArsD, which are members of the
ars operon, are responsible for the biosynthesis of C(22)-C(26) fatty acids
from malonyl-CoA. In vivo and in vitro reconstitution of
phenolic lipid synthesis systems with the Ars enzymes suggested that the
C(22)-C(26) fatty acids produced by ArsA and ArsD remained attached to the
ACP domain of ArsA and were transferred hand-to-hand to the active-site
cysteine residues of ArsB and ArsC. The type III PKSs then used the
fatty acids as starter substrates and carried out two or three extensions
with malonyl-CoA to yield the phenolic lipids. The phenolic lipids in A.
vinelandii were thus found to be synthesized solely from malonyl-CoA by
the four members of the ars operon. This is the first demonstration that a
type I FAS interacts directly with a type III PKS through substrate
transfer.
Zurück zum Text
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Kozubek, A., Pietr, S., Czerwonka, A., 1996.
Alkylresorcinols are abundant lipid components in different strains of Azotobacter chroococcum and
Pseudomonas spp. Journal of
Bacteriology 178, 4027-4030.
The occurrence of various
amounts of 5-n-alkyl resorcinols was shown in lipids extracted from 14
bacterial strains of Azotobacter chroococcum as well as from
strains of Pseudomonas aureofaciens, P. chlororapsis, and P.
fluorescens. The amount of alkylresorcinols found varied from 2.3 to
56.2 microg/mg (dry weight) of cells in A. chroococum and from
0.2 to 0.8 microg/mg (dry weight) of cells in Pseudomonas spp.
Strains of both genera produce saturated homologs with C13 to C27 side
chains. C19, C21, and C23 homologs are predominant in and characteristic
for A. chroococum strains, the C15 homolog is predominant in and
characteristic for P. chlororapsis and P. fluorescens, and
the C17 homolog is predominant in and characteristic for P.
aureofaciens. The presence of 5-n-(2-ketoalkyl)resorcinols, not
previously observed, was demonstrated in lipids isolated from the cells
of A. chroococum Az5.
Zurück zum Text
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Kozubek, A., Zarnowski, R., Stasiuk, M.,
Gubernator, J., 2001. Natural amphiphilic phenols as bioactive compounds.
Cellular & Molecular Biology Letters 6, 351-355.
Short review, no abstract.
Zurück zum Text
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Reusch, R. N., Sadoff, H. L., 1979.
5-n-Alkylresorcinols from encysting Azotobacter vinelandii:
isolation and characterization. Journal of Bacteriology 139, 448-453.
Azotobacter vinelandii was found to form novel lipid compounds when
encystment was initiated by 0.2% beta-hydroxybutyrate. An examination of
these compounds led to the isolation and characterization of
5-n-heneicosylresorcinol, 5-n-tricosylresorcinol, and their galactoside
derivatives.
Zurück zum Text
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Reusch, R. N., Sadoff, H. L., 1983. Novel lipid
components of the Azotobacter vinelandii cyst membrane. Nature 302,
268-270.
Phospholipids are ubiquitous components of biological membranes. In the
vegetative cells of Azotobacter vinelandii, a Gram-negative
free-living aerobic soil bacterium, the membrane lipids are
phospholipids with polar head group and fatty acyl compositions similar
to those of Escherichia coli. We report here that when A.
vinelandii differentiates to form metabolically dormant cysts, the
phospholipids in the membranes are replaced by a family of
5-n-alkylresorcinols and 6-n-alkylpyrones. These novel amphiphilic
lipids form a unique membrane matrix which may contribute to the
physiology and desiccation resistance of the cyst.
Zurück zum
Text
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Zarnowski, R., Suzuki, Y., Zarnowska, E. D.,
Esumi, Y., Kozubek, A., Pietr, S. J., 2004. 5-n-alkylresorcinols from the
nitrogen-fixing soil bacterium Azotobacter chroococcum Az12.
Zeitschrift für Naturforschung 59c, 318-320.
No Abstract
Zurück zum Text
Zum Seitenanfang
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