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(Last modification:
27. November 2009)
Type III Polyketide Synthases
(PKS) in Bacteria
Pyrone Type Ring-Folding
Orphan
PKS in Bacteria
1.
Mycobacterium
tuberculosis
(Saxena et al.,
2003,
Sankaranarayanan et al., 2004)
The
two proteins, PKS18 and PKS11 are fascinating examples;
unusual in some ways in the family of CHS-related proteins is the preference for
very long-chain fatty acid CoA-esters (but look at the fungal type III from
Neurospora crassa: similar substrate preferences:
more...). Otherwise there is not much unusual about
the enzymes: they carry out two or three condensation reactions, and fold the
linear polyketides into pyrones; essentially the same as found as derailment
products with plant polyketide synthases. It is interesting that there is alreay
a 3D-structure of the protein: it tells us much about the large substrate
binding tunnel (Sankaranarayanan et al., 2004).
Reactions of
the type III PKS in Mycobacterium tuberculosis.
It is also remarkable that the two proteins
PKS18 and PKS11 are localized in
so different positions of the relationship tree, although the functions are very
similar. However, maybe this is a hint that such large active site cavities are
possibly a typical property of bacterial type III PKS: remember that
the PhlD from Pseudomonas fluorescens also accepted large starter
CoA-esters and synthesized similar products, although the physiological
substrate is malonyl-CoA (more...).
Essentially the same was reported for RppA from Streptomyces (more...).
It really is unfortunate that these products or their
derivatives apparently have not been found in Mycobacteria: so what is their
function? Are the long-chain fatty acid CoA-esters really the physiological
substrates? Based on the presently available evidence, these proteins should be
classified as 'Orphan
PKS'.
Mycobacteria also are a nice example of cross-talk
between fatty acid biosynthesis and polyketide synthases (PKS), see for example
Trivedi et al., 2004;
Arora et al., 2005;
Gokhale et al., 2007;
Arora et al., 2009. An
interesting aspect is that fatty acids are activated first by adenylation, and
then the activated acyl chain is converted to a CoA-ester that is used for PKS
reactions.
In this context you should also look at
DIF-1
biosynthesis in Dictyostelium
discoideum: a type III PKS covalently coupled to a fatty acid
biosynthesis system: more...
2.
Bacillus subtilis
(Nakano et
al., 2009)
These results just came out, May 2009.
It looks like another example of a bacterial type III PKS that uses long-chain
fatty acid CoA-esters as substrates and in vivo carries out two
condensations to form triketide pyrones, just as shown above for Mycobacterium. In vitro
there are additional products: tetraketide pyrones and alkylresorcinols (i.e.
an STS-type ring-folding after three condensations). The enzyme up to this point
is clearly an orphan PKS because deletion of the operon did not reveal
any phenotypic changes, and the in vivo function remains unknown.
Links
to other examples of orphan type III PKS
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Go to 'orphan'
overview
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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Links to bacterial type III PKS
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References
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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);
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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.
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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
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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.
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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.
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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.
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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.
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