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(Last modification:
27. November 2009)
Typ III Polyketidsynthasen (PKS) in Bakterien
Pyron Typ Ringfaltung
Orphan
PKS in Bakterien
2.
Bacillus subtilis
1.
Mycobacterium tuberculosis
(Saxena
et al., 2003,
Sankaranarayanan et al., 2004)
Dies ist ein faszinierendes
Beispiel, und ungewöhnlich (s. jedoch die Eigenschaften von PhlD aus
Pseudomonas fluorescens ! Mehr...) in der Familie der CHS-verwandten
Proteine: Die eindeutige Bevorzugung von langkettigen Fettsäuren als Starter CoA-Ester! Ein neueres Beispiel mit fast den gleichen Präferenzen (aber anderen
Produkten!) ist die Typ III PKS aus Neurospora crassa:
Mehr.... Ansonsten sind die Reaktionen nicht sehr ungewöhnlich: Die Enzyme
führen zwei und drei Kondensations-Reaktionen durch und falten die linearen
Polyketide zu Pyronen; essentiell wie sie als Nebenprodukte bei vielen
CHS-verwandten Proteinen in Pflanzen gefunden wurden.
Interessant ist, dass es hier auch bereits eine
Kristall-Struktur gibt! Sehr interessant wie die langen Substrate am Enzym
untergebracht werden (Sankaranarayanan et al., 2004)!
.
Reaktionen
der Typ III PKS in Mycobacterium tuberculosis.
Bemerkenswert ist auch, dass die beiden
Proteine
PKS18 and PKS11 an so verschiedenen
Positionen im Verwandtschaftsbaum lokalisiert sind. Dies könnte jedoch ein
Hinweis sein, dass die sehr grossen aktiven Taschen von bakteriellen Typ III PKS eine gemeinsame Eigenschaft von sonst ganz verschiedenen Proteinen
sein könnten: Ein Beispiel ist das PhlD aus
Pseudomonas fluorescens,
welches auch sehr grosse Substrate akzeptiert und Pyrone bildet, obwohl das
physiologische Substrat Malonyl-CoA ist und damit eine CHS-Typ Ringfaltung
durchgeführt wird (mehr...). Und
ähnliches wurde für
RppA from Streptomyces berichtet (mehr...).
Sehr schade ist, dass die bekannten Produkte oder Derivate
davon anscheinend bisher nicht in Mycobacterien gefunden wurden. Also
automatisch die Frage: Sind dies wirklich die physiologischen Substrate? Und was
machen die Proteine denn nun wirklich? Nach dem jetzigen Kenntnisstand sollten
sie als 'Orphan
PKS' klassifiziert werden.
Mycobacterien sind auch ein schönes Beispiel für
"cross-talk" zwischen Fettsäure-Biosynthese und Polyketidsynthasen (PKS),
siehe zum Beispiel
Trivedi
et al., 2004; Arora et al., 2005;
Gokhale et al., 2007;
Arora et al., 2009. Ein
interessanter Aspekt ist, dass die Fettsäuren zunächst durch Adenylierung
aktiviert werden, und dann wird die aktivierte Acyl-Kette in einen CoA-Ester für
die PKS-Reaktionen umgewandelt. In diesem Zusammenhang sollten Sie sich auch die
DIF-1 Biosynthese in
Dictyostelium
discoideum ansehen: Eine Typ III PKS, die kovalent mit einem
Fettsäure-Biosynthese-System verknüpft ist:
Mehr...
Zum Seitenanfang
2.
Bacillus subtilis
(Nakano et
al., 2009)
Diese Ergebnisse sind ganz neu, Mai
2009, im Druck: August 2009. Diese Typ III PKS aus Bacillus subtilis ist ein weiteres Beispiel
für Enzyme, welche langkettige Fettsäure-CoA-Ester als Substrate verwenden und
in vivo zwei Kondensationen durchführen, mit den entsprechenden Triketid-Pyronen als Produkten, wie oben für Mycobacterium gezeigt. In vitro, mit
rekombinanten Proteinen, gibt es auch weitere Produkte, solche aus drei
Kondensationsreaktionen, wie Tetraketid-Pyrone und Alkylresorcinole (also
Produkte aus STS-Typ Ringfaltungen). Bis jetzt muss das Enzym wohl als "Orphan"
bezeichnet werden: Die Deletion des Gens führte zu keiner erkennbaren
Veränderung der Bakterien. Die Funktion in vivo bleibt also noch unklar.
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Übrigens
gibt es Typ III PKS mit Substrat-Präferenzen für langkettige CoA-Estern
anscheinend häufig in Pflanzen und in Bakterien. Beispiele sind:
-
-
-
-
Alkylresorcinole
und
langkettige Pyrone in dem Bakterium Azotobacter vinelandii:
Mehr...
-
Alkylresorcinol-
Streptomyces
griseus: Mehr...
-
Pyronsynthasen in den Bakterien
Mycobacterium tuberculosis und
Bacillus subtilis: Mehr...
-
CsyA: Pyronsynthasen
in dem Pilz Aspergillus oryzae:
Mehr...
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Links zu anderen Beispielen von 'Orphan PKS'
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Links zu bakteriellen Typ III PKS
Zum Seitenanfang
Zitate
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Saxena, P., Yadav, G., Mohanty, D., Gokhale, R. S.:
A new family of type III polyketide synthases in Mycobacterium tuberculosis.
Journal of Biological Chemistry 278, 44780-44790 (2003).
The Mycobacterium
tuberculosis genome has revealed a remarkable array of polyketide
synthases (PKSs); however, no polyketide product has been isolated thus far.
Most of the PKS genes have been implicated in the biosynthesis of complex
lipids. We report here the characterization of two novel type III PKSs from
M. tuberculosis that are involved in the biosynthesis of long-chain
alpha-pyrones. Measurement of steady-state kinetic parameters demonstrated
that the catalytic efficiency of PKS18 protein was severalfold higher for
long-chain acyl-coenzyme A substrates as compared with the small-chain
precursors. The specificity of PKS18 and PKS11 proteins toward long-chain
aliphatic acyl-coenzyme A (C12 to C20) substrates is unprecedented in the
chalcone synthase (CHS) family of condensing enzymes. Based on comparative
modeling studies, we propose that these proteins might have evolved by
fusing the catalytic machinery of CHS and beta-ketoacyl synthases, the two
evolutionarily related members with conserved thiolase fold. The mechanistic
and structural importance of several active site residues, as predicted by
our structural model, was investigated by performing site-directed
mutagenesis. The functional identification of diverse catalytic activity in
mycobacterial type III PKSs provide a fascinating example of metabolite
divergence in CHS-like proteins.
Protein accessions:
Pks18 =
YP_177803 (there are
several more entries with the same protein sequence);
Pks11 =
NP_216181 (several very
similar sequences from other Mycobacteria); Pks10
= NP_216176 (several very similar sequences
from other Mycobacteria);
Zurück zum Text
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Sankaranarayanan, R., Saxena, P., Marathe, U. B., Gokhale, R.
S., Shanmugam, V. M., Rukmini, R.:
A novel tunnel in mycobacterial type III polyketide synthase reveals the
structural basis for generating diverse metabolites.
Nature Structural and Molecular Biology 11, 894-900 (2004).
The superfamily of plant
and bacterial type III polyketide synthases (PKSs) produces diverse
metabolites with distinct biological functions. PKS18, a type III PKS from
Mycobacterium tuberculosis, displays an unusual broad specificity for
aliphatic long-chain acyl-coenzyme A (acyl-CoA) starter units (C(6)-C(20))
to produce tri- and tetraketide pyrones. The crystal structure of PKS18
reveals a 20 A substrate binding tunnel, hitherto unidentified in this
superfamily of enzymes. This remarkable tunnel extends from the active site
to the surface of the protein and is primarily generated by subtle changes
of backbone dihedral angles in the core of the protein. Mutagenic studies
combined with structure determination provide molecular insights into the
structural elements that contribute to the chain length specificity of the
enzyme. This first bacterial type III PKS structure underlines a fascinating
example of the way in which subtle changes in protein architecture can
generate metabolite diversity in nature.
Zurück zum Text
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Nakano, C., Ozawa, H., Akanuma, G., Funa, N.,
Horinouchi, S., 2009. Biosynthesis of aliphatic polyketides by type III
polyketide synthase and methyltransferase in Bacillus subtilis.
Journal of Bacteriology
191, 4916-4923.
Type III polyketide synthases (PKSs) synthesize a variety of aromatic
polyketides in plants, fungi and bacteria. The bacterial genome projects
predicted that probable type III PKS genes are distributed in a wide variety
of gram-positive and negative bacteria. The gram-positive model
microorganism Bacillus subtilis contained the bcsA-ypbQ operon, which
appeared to encode a type III PKS and a methyltransferase, respectively.
Here we report the characterization of bcsA [renamed bpsA (Bacillus pyrone
synthase) on the basis of its function] and ypbQ that are involved in the
biosynthesis of aliphatic polyketides. In vivo analysis demonstrated
that BpsA was a type III PKS catalyzing the synthesis of triketide pyrones
from long-chain fatty acyl CoA thioesters as starter substrates and
malonyl-CoA as an extender substrate, and YpbQ was a methyltransferase
acting on the triketide pyrones to yield alkylpyrone methyl ethers. YpbQ was
thus named BpsB because of the functional relatedness to BpsA. In vitro
analysis with a histidine-tagged BcsA revealed that it used broad starter
substrates and produced not only triketide pyrones but also tetraketide
pyrones and alkylresorcinols. Although the aliphatic polyketides were
expected to localize in the membrane and play some role in modulating
rigidity and properties of the membrane, no detectable phenotypic changes
were observed for a B. subtilis mutant containing a whole deletion of
the bcsA-ypbQ operon.
Accession of protein:
NP_390087
Zurück
-
Arora, P., Goyal, A., Natarajan, V. T., Rajakumara,
E., Verma, P., Gupta, R., Yousuf, M., Trivedi, O. A., Mohanty, D., Tyagi,
A., Sankaranarayanan, R., Gokhale, R. S., 2009. Mechanistic and functional
insights into fatty acid activation in Mycobacterium tuberculosis.
Nature Chemical Biology 5, 166-173.
The recent discovery of fatty acyl-AMP ligases (FAALs) in
Mycobacterium tuberculosis (Mtb) provided a new perspective of fatty
acid activation. These proteins convert fatty acids to the corresponding
adenylates, which are intermediates of acyl-CoA-synthesizing fatty acyl-CoA
ligases (FACLs). Presently, it is not evident how obligate pathogens such as
Mtb have evolved such new themes of functional versatility and whether the
activation of fatty acids to acyladenylates could indeed be a general
mechanism. Here, based on elucidation of the first structure of an FAAL
protein and by generating loss-of-function and gain-of-function mutants that
interconvert FAAL and FACL activities, we demonstrate that an insertion
motif dictates formation of acyladenylate. Because FAALs in Mtb are crucial
nodes in the biosynthetic network of virulent lipids, inhibitors directed
against these proteins provide a unique multipronged approach to
simultaneously disrupting several pathways.
Zurück zum Text
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Arora, P., Vats, A., Saxena, P., Mohanty, D.,
Gokhale, R. S., 2005. Promiscuous fatty acyl CoA ligases produce acyl-CoA
and acyl-SNAC precursors for polyketide biosynthesis. Journal of the
American Chemical Society 127, 9388-9389.
The study of bioactive natural products has undergone rapid
advancement with the cloning and sequencing of large number of gene clusters
and the concurrent progress to manipulate complex biosynthetic systems in
heterologous hosts. The genetic reconstitution necessitates that the
heterologous hosts possess substrate pools that could be coordinately
supplied for biosynthesis. Polyketide synthases (PKS) utilize acyl-coenzyme
A (CoA) precursors and synthesize polyketides by repetitive decarboxylative
condensations. Here we show that acyl-CoA ligases, which belong to a large
family of acyl-activating enzymes, possess potential to produce varied
starter CoA precursors that could be utilized in polyketide biosynthesis.
Incidentally, such protein domains have been recognized in several PKS and
nonribosomal peptide synthetase gene clusters. Our studies with
mycobacterial fatty acyl-CoA ligases (FACLs) show remarkable tolerance to
activate a variety of fatty acids that contain modifications at alpha, beta,
omega, and omega-nu positions. This substrate flexibility extends further
such that these proteins also efficiently utilize N-acetyl cysteamine, the
shorter acceptor terminal portion of CoASH, to produce acyl-SNACs. We show
that the in situ generated acyl-CoAs and acyl-SNACs could be channeled to
type I and type III PKS systems to produce new metabolites. Together, the
promiscuous activity of FACL and PKSs provides new opportunities to expand
the repertoire of natural products.
Zurück zum Text
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Gokhale, R. S., Saxena, P., Chopra, T., Mohanty,
D., 2007. Versatile polyketide enzymatic machinery for the biosynthesis of
complex mycobacterial lipids. Natural Product Reports 24, 267-277.
The cell envelope of Mycobacterium tuberculosis (Mtb) is a
treasure house of a variety of biologically active molecules with
fascinating architectures. The decoding of the genetic blueprint of Mtb in
recent years has provided the impetus for dissecting the metabolic pathways
involved in the biosynthesis of lipidic metabolites. The focus of the
Highlight is to emphasize the functional role of polyketide synthase (PKS)
proteins in the biosynthesis of complex mycobacterial lipids. The catalytic
as well as mechanistic versatility of PKSs in generating metabolic diversity
and the significance of recently discovered fatty acyl-AMP ligases in
establishing "biochemical crosstalk" between fatty acid synthases (FASs) and
PKSs is described. The phenotypic heterogeneity and remodeling of the
mycobacterial cell wall in its aetiopathogenesis is discussed.
Zurück zum Text
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Trivedi,
O. A., Arora, P., Sridharan, V., Tickoo, R., Mohanty, D., Gokhale, R. S.,
2004. Enzymic activation and transfer of fatty acids as acyl-adenylates in
mycobacteria. Nature 428, 441-445.
The metabolic repertoire in nature is augmented by generating hybrid
metabolites from a limited set of gene products. In mycobacteria, several
unique complex lipids are produced by the combined action of fatty acid
synthases and polyketide synthases (PKSs), although it is not clear how the
covalently sequestered biosynthetic intermediates are transferred from one
enzymatic complex to another. Here we show that some of the 36 annotated
fadD genes, located adjacent to the PKS genes in the Mycobacterium
tuberculosis genome, constitute a new class of long-chain fatty acyl-AMP
ligases (FAALs). These proteins activate long-chain fatty acids as
acyl-adenylates, which are then transferred to the multifunctional PKSs for
further chain extension. This mode of activation and transfer of fatty acids
is contrary to the previously described universal mechanism involving the
formation of acyl-coenzyme A thioesters. Similar mechanisms may operate in
the biosynthesis of other lipid-containing metabolites and could have
implications in engineering novel hybrid products.
Zurück zum Text
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