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(Last modification:
10. April 2010)
'Orphan'
PKS: Eine Pilz Typ III PKS
mit bis zu vier Kondensationsreaktionen:
ORAS aus Neurospora crassa
(Wirklich eine
'Orphan' PKS? Es gibt neuere Vermutungen, dass die physiologische Rolle die
Biosynthese von lang-kettigen Resorcinolen sein könnte:
Goyal et al., 2008)
Neurospora crassa
enthält anscheinend ein einziges Gen für eine Typ III PKS. Die Funktion des
Proteins wurde kürzlich untersucht, mit ganz interessanten Ergebnissen (Funa
et al., 2007). Das rekombinante Protein hatte keine Aktivität mit
4-Coumaroyl-CoA, dem für Chalconsynthase (CHS) und Stilbensynthase (STS)
typischen Substrat. Es akzeptierte jedoch alle Arten von aliphatischen
CoA-Estern, mit Kettenlängen von vier bis zwanzig C-Atomen, und produzierte mit
ihnen eine erstaunliche Anzahl von Produkten. Einige werden in der Abbildung
zusammengefasst.
Interessant: Nicht alle Produkte wurden
von allen Substraten synthetisiert; die Art der Produkte war stark abhängig von
der Kettenlänge der Substrate:
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Triketid-Pyrone wurden aus C4, C6,
und C8 Startern synthetisiert,
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Triketid-Pyrone, begleitet von
kleinen Mengen Tetraketid-Pyronen und
Tetraketid-Resorcinolen wurden aus C10, C12, und C14 Startern
aufgebaut,
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Tetraketid- und Pentaketid-Resorcylsäuren wurden
aus
C16, C18, und C20 Startern synthetisiert.
Mit den grössten
Substraten waren die Pentaketid-Resorcylsäuren die einzigen Produkte in kurzen
Inkubationen, und erst längere Inkubationen führten zu den entsprechenden
Resorcinolen. Dies war ein guter Hinweis darauf, dass die Decarboxylierung zu
den Resorcinolen nicht-enzymatisch mit den unstabilen Resorcylsäuren stattfand.
Dementsprechend wurde das Enzym bezeichnet als
2'-Oxoalkylresorcylsäure-Synthase
(englisch:
2'-oxoalkylresorcylic
acid synthase =
ORAS).
Diese namensgebende Aktivität scheint wirklich ein 'First' zu sein: Vier
Kondensationen und dann ein Ringschluss zur Resorcylsäure. Es ist wirklich die
Kombination 'vier und Ringschluss-Typ'. Der gleiche Ringschluss-Typ, aber mit nur drei
Kondensationen, findet sich auch bei Typ III PKS aus Pflanzen, z.B. bei
der Synthese der Stilbencarboxylate (Hydrangea macrophylla
, und
Lebermoos
Marchantia
polymorpha), und postuliert wird er in der Tetrahydrocannabinol-Synthese
im Hanf (Cannabis
sativa): Mehr....
Und was ist die physiologische
Funktion dieses Typ III Enzyms aus Pilzen? Ein Rätsel, jedenfalls bis jetzt, und
einige zusätzliche Experimente brachten nicht viel Aufschluss. RT-PCR Analysen
der Expression deuteten daraufhin, dass das Gen immer aktiv ist. Die
Zerstörung/Inaktivierung des Gens ergab keinen identifizierbaren Phänotyp, und
man fand überhaupt keinen Unterschied zwischen Mutante und Wildtyp, und
tatsächlich hat man auch aus der Analyse der Naturstoffe keine Ahnung, für
welche Biosynthese dieses Enzyms denn wohl zuständig sein könnte. Das Problem
ist hier wirklich: Wonach sollte man denn suchen? Das Enzym kann viel zu viel!
Ausserdem ist gut möglich, dass Intermediate oder das Produkt der PKS durch
zusätzliche Reaktionen modifiziert werden: Das macht es auch nicht gerade
leichter, da dies die Zahl der möglichen Produkte weiter erhöht. Unter der
Annahme, dass dieses Protein tatsächlich etwas in vivo macht (auch das
muss nicht unbedingt so sein!): Man wird wohl erst eine detaillierte Analyse der
Naturstoffe durchführen müssen. Frage: Wer hat denn dafür Zeit und Geld, wenn
man noch nicht einmal eine Idee hat, wonach zu suchen ist?
Das Enzym sollte also nach bisherigem Kenntnisstand als 'Orphan
PKS' bezeichnet werden.
An interesting question is whether this
enzyme
is likely to use the
aldol switch mechanism discovered with the
stilbene synthase (STS) from
Scots pine (Pinus sylvestris):
more...
Die 3D-Struktur
More recent
work revealed insights into the 3D-structure of this enzyme:
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Goyal et al. (June 2008)
It is noteworthy that their assays
with the recombinant protein revealed tetraketide-derived resorcinols as the
major products of the enzyme reaction with very long-chain fatty acid CoA-esters
(i.e. 18, 20, 22, or 24 carbons), not
the pentaketide-derived 2'-oxoalkylresorcylic acids described in the first
characterization of the enzyme (Funa et al., 2007, see above).
Based on these
and other results the authors propose that the enzyme is an alkylresorcinol
synthase in vivo, i.e. carries out only three condensations. In this context it is noteworthy that at least one
fungus, Fusarium culmorum, has indeed been shown to secrete
5-n-alkylresorcinols (Zarnowski
et al., 2000). Type III alkylresorcinol synthases have also been described
in Physcomitrella patens (biological function unknown,
also an 'orphan PKS') and in the
biosynthesis of sorgoleone in Sorghum bicolor:
more..., and also bacteria do
contain such enzymes (Azotobacter vinelandii:
more...).
The
3D-structure (resolution 2.58
Å)
revealed a long hydrophobic tunnel most likely responsible for its
specificity for long-chain fatty acids; this resembles the substrate binding
tunnel in the structure of PKS18, a mycobacterial type III PKS (Sankaranarayanan
et al., 2004; Rukmini et al.,
2004,
more...). The structure
suggested several specific residues lining the active site tunnel as
responsible, and the authors picked one of these for mutagenesis studies:
Ser186. They speculated that its mutation to the much bulkier Phe might block
the tunnel, and thus would change the substrate preference to much shorter fatty
acid CoA-esters (this was the result
with the analogous mutation in the mycobacterial PKS18; see:
Sankaranarayanan et al., 2004). Somewhat surprisingly, the fungal enzyme
reacted differently: it still accepted long-chain acyl-CoAs, but it could no
longer produce resorcinols; the products were primarily pyrones. The interesting
conclusion is therefore that not the substrate acceptance, but the cyclization
to the end product was affected by this mutation. A more detailed inspection of
the mutant revealed that the introduced Phe could be modelled in three different rotamers,
and all of them were sterically feasible. One of these actually predicted a
position in which it would not block the tunnel, because a Ser in position 340
would permit this rotamer. Therefore the authors mutagenized Ser340 to Leu, thus
hindering with a larger side-chain this particular positioning of the Phe side-chain. As predicted, the
double mutant was no longer capable of activity with long-chain acyl-CoA esters.
The mechanism of the STS-type ring-folding to the resorcinols remains to be
explained. The available results suggests, however, that a hydrogen-bonding
network like observed with the STS from Scots pine (Pinus sylvestris) (more...)
was not present, suggesting that there is an alternative mechanism for aldol
condensation in the fungal enzyme.
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Rubin-Pitel
et al. (October 2008)
This
publication came only recently to my attention. It describes the analysis of
the crystal structure to 1.75 Å resolution, and, based on that, the analysis
of a few potentially interesting mutants:
-
They
confirmed that the Cys predicted for the active site is actually Cys152:
the mutant containing Thr in this position was totally inactive,
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They
analyzed the function of Cys120. This was interesting because this
residue is in the position analogous to Thr132 in the stilbene synthase
(STS) from Pinus sylvestris. The crystal structure of that enzyme had
shown that the positioning of this residue is critical to the formation of
the hydrogen-bonding network involved in the aldol condensation to the
resorcinol (more…). However, the
analysis of an ORAS Cys120->Ser mutation showed that the overall activity of
the enzyme was diminished, but the resorcinol formation was not abolished.
This indicated that the ORAS mechanism for resorcinol formation is different
from that in the P. sylvestris STS. Another clear difference between
STS and ORAS is, of course, that one of the ORAS products is a resorcylic
acid which is then decarboxylated to the alkyl resorcinol: no such
carboxylated product was ever detected with plant enzymes carrying out an
aldol condensations, e.g. STS and bibenzyl synthase (more…),
biphenyl synthase (more…),
the type III PKS in sorgoleone biosynthesis (more…),
or an alkylresorcinol synthase from Physcomitrella patens (more…).
In addition, a study specifically directed at this question had shown with a
STS that decarboxylation occurred earlier than aromatization (Shibuya
et al., 2002).
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Phe252
in ORAS corresponds to Gly256 in the CHS from Medicago sativa. This
Gly is highly conserved in CHS and plays an important role in defining the
active site cavity. This was demonstrated very convincingly in the
mechanistic analysis of the pyrone synthase (2-PS) from Gerbera
hybrida: the mutagenesis of Gly256 in the Medicago sativa CHS to
the Leu of 2PS was one of the only three modifications that converted the
CHS into a pyrone synthase capable of accepting only small substrates and
carrying out only two condensation reactions (more…
).
Phe252 in ORAS is poised to serve as a steric
gate to regulate product specificity. Substitution of Phe252 to Gly in the
ORAS enzyme altered the product profile for long-chain acyl-CoA substrates
(C16 or larger), and specifically disrupted the production of pentaketide
resorcinol and resorcylic acid products; there was a clear shift to pyrone
products. In summary, it appears that Phe252 plays a role in stabilizing the
extended linear polyketide chain such that resorcinol production is favoured
against pyrone formation. The structural analysis of this mutant (2.1
Å resolution) essentially confirmed these conclusions.
Please look at the publication for a more detailed
description; there is lots of interesting results and discussions!
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In the
discussion the authors make some interesting remarks concerning the
preceding publication by Goyal et al. (2008). Their model of the fatty acid
binding site was based on the structure of the bacterial Mycobacterium
tuberculosis PKS18 bound to myristic acid, but according to the work
discussed here this did not provide a correct picture, and thus led to
misleading predictions; in their opinion it was not surprising that the
Ser186 mutant had such unexpected consequences.
Zum Seitenanfang
Links zu anderen Beispielen von 'Orphan PKS'
Zum Seitenanfang
Zitate
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Funa, N., Awakawa, T., Horinouchi, S., 2007.
Pentaketide resorcylic acid synthesis by type III polyketide synthase from
Neurospora crassa. Journal of Biological Chemistry 282, 14476-14481.
Type III polyketide synthases (PKSs) are responsible for aromatic
polyketide synthesis in plants and bacteria. Genome analysis of
filamentous fungi has predicted the presence of fungal type III PKSs,
although none have thus far been functionally characterized. In the
genome of Neurospora crassa, a single open reading frame,
NCU04801.1, annotated as a type III PKS was found. In this report, we
demonstrate that NCU04801.1 is a novel type III PKS catalyzing the
synthesis of pentaketide alkylresorcylic acids. NCU04801.1, hence named
2'-oxoalkylresorcylic acid synthase (ORAS), preferred stearoyl-CoA as a
starter substrate and condensed four molecules of malonyl-CoA to give a
pentaketide intermediate. For ORAS to yield pentaketide alkylresorcylic
acids, aldol condensation and aromatization of the intermediate, which
is still attached to the enzyme, are presumably followed by hydrolysis
for release of the product as a resorcylic acid. ORAS is the first type
III PKS that synthesizes pentaketide resorcylic acids.
Protein accession and link to
sequence: XP_960427
Zurück zum Text
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Goyal, A., Saxena, P., Rahman, A.,
Singh, P. K., Kasbekar, D. P., Gokhale, R. S., Sankaranarayanan, R., 2008.
Structural insights into biosynthesis of resorcinolic lipids by a type III
polyketide synthase in Neurospora crassa. Journal of Structural
Biology
162, 411-21.
Microbial type III polyketide synthases (PKSs) have revealed
remarkable mechanistic as well as functional versatility. Recently, a
type III PKS homolog from Azotobacter has been implicated in the
biosynthesis of resorcinolic lipids, thus adding a new functional
significance to this class of proteins. Here, we report the structural
and mutational investigations of a novel type III PKS protein from
Neurospora crassa involved in the biosynthesis of resorcinolic
metabolites by utilizing long chain fatty acyl-CoAs. The structure
revealed a long hydrophobic tunnel responsible for its fatty acyl chain
length specificity resembling that of PKS18, a mycobacterial type III
PKS. Structure-based mutational studies to block the tunnel not only
altered the fatty acyl chain specificity but also resulted in change of
cyclization pattern affecting the product profile. This first structural
characterization of a resorcinolic lipid synthase provides insights into
the coordinated functioning of cyclization and a substrate-binding
pocket, which shows mechanistic intricacy underlying type III PKS
catalysis.
Zurück zum Text
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Rubin-Pitel, S. B., Zhang, H., Vu, T.,
Brunzelle, J. S., Zhao, H., Nair, S. K., 2008.
Distinct structural elements
dictate the specificity of the type III pentaketide synthase from
Neurospora crassa. Chemistry and Biology 15, 1079-1090.
The fungal type III polyketide synthase 2'-oxoalkylresorcylic acid
synthase (ORAS) primes with a range of acyl-Coenzyme A thioesters
(C4-C20) and extends using malonyl-Coenzyme A to produce pyrones,
resorcinols, and resorcylic acids. To gain insight into this unusual
substrate specificity and product profile, we have determined the
crystal structures of ORAS to 1.75 Å resolution, the Phe252->Gly
site-directed mutant to 2.1 Å resolution, and a binary complex of ORAS
with eicosanoic acid to 2.0 Å resolution. The structures reveal a
distinct rearrangement of structural elements near the active site that
allows accommodation of long-chain fatty acid esters and a reorientation
of the gating mechanism that controls cyclization and polyketide chain
length. The roles of these structural elements are further elucidated by
characterization of various structure-based site-directed variants.
These studies establish an unexpected plasticity to the PKS fold,
unanticipated from structural studies of other members of this enzyme
family.
(Excellent work, but just be careful with one of the methods: a
concentration of 100 mM malonyl-CoA in the kinetic analysis is
probably a typo).
Return
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Rukmini, R., Shanmugam, V. M., Saxena, P.,
Gokhale, R. S., Sankaranarayanan, R., 2004. Crystallization and preliminary
X-ray crystallographic investigations of an unusual type III polyketide
synthase PKS18 from Mycobacterium tuberculosis. Acta Crystallography
Section D Biological Crystallography 60, 749-751.
The
biosynthetic machinery of polyketide synthases involves various
sequential enzymatic reactions, such as initiation, elongation and
cyclization, to produce polyketides. PKS18 protein from Mycobacterium
tuberculosis belongs to the type III polyketide synthase family and
displays an unusual starter-unit specificity to catalyze the formation
of alpha-pyrones. This enzyme uses malonyl-CoA to iteratively extend
long-chain aliphatic coenzyme A (C12 to C20) molecules, producing
triketide and tetraketide pyrone products. In order to aid in
understanding the structural basis of this long-chain specificity and to
further characterize the enzymatic mechanism of PKS18, the protein has
been crystallized. The crystal belongs to the triclinic space group P1,
with unit-cell parameters a = 59.9, b = 80.7, c = 99.6 A, alpha = 108.2,
beta = 93.0, gamma = 103.7 degrees.
Mehr...
Zurück zum Text
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Sankaranarayanan, R., Saxena, P.,
Marathe, U. B., Gokhale, R. S., Shanmugam, V. M., Rukmini, R., 2004. A
novel tunnel in mycobacterial type III polyketide synthase reveals the
structural basis for generating diverse metabolites. Nature Structural &
Molecular Biology 11, 894-900.
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.
Mehr...
Zurück zum Text
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Shibuya, M., Nishioka, M., Sankawa, U.,
Ebizuka, Y., 2002. Incorporation of three deuterium atoms excludes
intermediacy of stilbenecarboxylic acid in stilbene synthase reaction.
Tetrahedron Letters 43, 5071-5074.
To investigate the mechanism of stilbene synthase (STS) reaction, the
origin of the aromatic protons of resveratrol B-ring was examined using
STS from Arachis hypogaea expressed in Escherichia coli
and deuterated malonyl-CoA. The presence of resveratrol labeled with
three deuterium atoms was detected by GCMS analysis indicating
decarboxylation earlier than aromatization. i.e. exclusion of
intermediacy of stilbenecarboxylic acid in STS reaction.
Zurück
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Zarnowski, R., Lewicka, T., Pietr, S. J., 2000.
Production and secretion of 5-n-alkylresorcinols by Fusarium culmorum.
Zeitschrift für
Naturforschung 55c, 846-848.
Fusarium culmorum F1 was found to
produce and secrete into the culture medium several of
5-n-alkylresorcinols. The amount of resorcinolic lipids was 5.3 microg/g
and 0.19 microg/l in mycelium and in post-culture liquid, respectively.
First of all F. culmorum F1 produces saturated homologues with C15 to
C25 side chains. The extract from the medium contained only homologues
with shorter carbon chains (C13 to C17).
Zurück zum Text
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