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(Last
modification: 24. February 2010)
Seven
condensation reactions: Octaketide Synthase (OKS) in Anthrone Biosynthesis
(Abe
et al., 2005; Abe 2006; preliminary crystal structure: Morita et
al., 2007)
This was actually
quite unexpected: A chalcone synthase related PKS
from plants carrying out seven condensations! The protein encoded in a cDNA cloned from aloe (Aloe arborescens) shared 50-60% amino acid sequence
identity with other plant CHS-superfamily enzymes, but 90% with the
pentaketide synthase from the
same plant.
A recombinant enzyme
expressed in Escherichia coli used malonyl-CoA as starter and catalyzed seven condensations
with malonyl-CoA to yield the aromatic octaketides SEK4 and SEK4b, the longest
polyketides known to be synthesized by plant type III PKS. Acetyl-CoA, resulting from decarboxylation of
malonyl-CoA, was also accepted as a starter substrate, just as in the case of the
previously reported A. arborescens PCS (pentaketide
synthase), but not so efficiently as in the
case of R. palmatum ALS (aloesone
synthase, a heptaketide synthase).
The figure shows the reaction which
in vitro leads to SEK4/SEK4b. As noted above, the reaction can be
initiated with malonyl-CoA or acetyl-CoA, although malonyl-CoA appears to be
more efficient. The products are identical with those of the 'minimal' PKS (i.e.
the basic, simplest unit) of the complex biosynthetic machinery for formation of
the antibiotic actinorhodin in the bacterium Streptomyces coelicolor (Carreras
et al., 1996) (the basic monomeric subunit is also synthesized via seven
condensation reactions).
The aloe plant does not produce these substances, but anthrones and
anthraquinones that are probably derived from octaketides. It is therefore
tempting to speculate that the OKS is in vivo involved in the
biosynthesis of those plant natural products, and the figure below shows an example
proposed in a recent
review (Abe,
2006). It should be noted that the reaction to the anthrone should contain a
reduction step, as in the biosynthesis of
plumbagin
(-> hexaketide synthase) and other reactions discussed in that context
(more...). Like
in those cases,
it seems reasonable that the lack of such reaction with a specific intermediate
was the
reason for not getting the predicted product.
Like
PCS, the OKS showed considerable substrate
promiscuity. It also accepted aromatic CoA-esters (e.g. 4-coumaroyl-CoA, cinnamoyl-CoA, and
benzoyl-CoA) and long-chain aliphatic fatty acyl CoA esters (hexanoyl-CoA, octanoyl-CoA, decanoyl-CoA,
dodecanoyl-CoA, tetradecanoyl-CoA, hexadecanoyl-CoA, octadecanoyl-CoA, and eicosanoyl-CoA)
as starters, but in all cases is carried out only two (-> triketides) and three
(-> tetraketides) condensation reactions. Only the products from 4-coumaroyl-CoA
will be given here:
bisnoryangonin
and
CTAL (two and three condensations). These two are also typical derailment
reactions in the in vitro reactions of
chalcone and stilbene synthases.
Recent work
(Shi
et al., 2009a, Shi et al., 2009b) revealed even more surprising capacities of this enzyme,
at least in vitro: for example the formation of a hexaketide stilbene and a heptaketide
chalcone. The synthesis of the latter was dramatically increased in an OKS N222G
mutant, and this mutant also produced SEC15 from ten malonyl-CoA!
The figure is taken from the Abstract of the publication; I just added a few
explanations
A note of interest: Surprisingly even typical CHS can
carry out up to seven condensations with untypical substrates if they are mutagenized in certain positions
(Abe et al.,
2006): it was demonstrated
that the Ser338->Val mutant of Scutellaria baicalensis CHS produced the octaketides
SEK4/SEK4b from eight molecules of malonyl-CoA. The octaketide-forming activity
was dramatically increased in a CHS triple mutant (Thr197->Gly / Gly256->Leu / Ser338->Thr). The
functional conversion is probably based on steric changes in residues lining the active-site cavity.
Update 2009
(see also
aloesone synthase)
The same
group reported three more type III PKS from Aloe arborescens (Mizuuchi
et al., 2009):
-
PKS3 turned out to be a multifunctional enzyme: it is a heptaketide
synthase that presumably is involved in the biosynthesis of
aloesone:
more...
-
PKS4 and PKS5
are functionally identical with the octaketide synthase
(OKS) described here
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Links to
the pages: Enzymes with more than three condensations
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References
-
Mizuuchi,
Y., Shi, S.-P., Wanibuchi, K., Kojima, A., Morita, H., Noguchi, H., Abe, I.,
2009. Novel type III polyketide synthases from Aloe arborescens. FEBS
Journal 276, 2391-2401.
Aloe arborescens is a medicinal plant rich in aromatic polyketides,
such as pharmaceutically important aloenin (hexaketide), aloesin (heptaketide)
and barbaloin (octaketide). Three novel type III polyketide synthases (PKS3,
PKS4 and PKS5) were cloned and sequenced from the aloe plant by cDNA library
screening. The enzymes share 85-96% amino acid sequence identity with the
previously reported pentaketide chromone synthase and octaketide synthase.
Recombinant PKS4 and PKS5 expressed in Escherichia coli were
functionally identical to octaketide synthase, catalyzing the sequential
condensations of eight molecules of malonyl-CoA to produce octaketides SEK4.
SEK4b. As in the case of octaketide synthase, the enzymes are possibly
involved in the biosynthesis of the octaketide barbaloin. On the other hand,
PKS3 is a multifunctional enzyme that produces a heptaketide
aloesone (i.e. the aglycone of aloesin) as a major product from seven
molecules of malonyl-CoA. In addition, PKS3 also afforded a hexaketide
pyrone (i.e. the precursor of aloenin), a heptaketide
6-(2-acetyl-3,5-dihydroxybenzyl)-4-hydroxy-2-pyrone, a novel heptaketide
6-(2-(2,4-dihydroxy-6-methylphenyl)-2-oxoethyl)-4-hydroxy-2-pyrone and
octaketides SEK4/SEK4b. This is the first demonstration of the enzymatic
formation of the precursors of the pharmaceutically important aloesin and
aloenin by a wild-type PKS obtained from A. arborescens.
Interestingly, the aloesone-forming activity was maximum at 50 degree C, and
the novel heptaketide pyrone was non-enzymatically converted to aloesone. In
PKS3, the active-site residue 207, which is crucial for controlling the
polyketide chain length depending on the steric bulk of the side chain, is
uniquely substituted with Ala. Site-directed mutagenesis demonstrated that
the A207G mutant dominantly produced the octaketides SEK4 / SEK4b, whereas
the A207M mutant yielded a pentaketide 5,7-dihydroxy-2-methylchromone.
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Shi, S. P., Wanibuchi, K., Morita, H., Endo, K.,
Noguchi, H., Abe, I., 2009a. Enzymatic formation of unnatural novel chalcone,
stilbene, and benzophenone scaffolds by plant type III polyketide synthase.
Organic Letters 11, 551-554.
A C(19) hexaketide stilbene and a C(21) heptaketide chalcone were
synthesized by Aloe arborescens octaketide synthase (OKS), a
plant-specific type III polyketide synthase (PKS). Remarkably, the C(21)
chalcone-forming activity was dramatically increased in a structure-guided
OKS N222G mutant that produces a C(20) decaketide SEK15 from 10 molecules of
malonyl-CoA. The findings suggested further strategies for production of
unnatural polyketides by combination of the precursor-directed biosynthesis
and the structure-guided engineering of type III PKS.
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Shi,
S.-P., Morita, H., Wanibuchi, K., Noguchi, H., Abe, I., 2009b. Enzymatic
formation of unnatural novel polyketide scaffolds by plant-specific type III
polyketide
synthase. Tetrahedron Letters 50,
2150-2153.
The catalytic potential of octaketide synthase (OKS), a plant-specific type
III polyketide synthase (PKS) from Aloe arborescens, was investigated
by phenylacetyl-CoA and benzoyl-CoA as starter substrates. As a result, a
novel C16 pentaketide coumarin was produced from phenylacetyl-CoA, whereas
benzoyl-CoA was not a good substrate of OKS. Remarkably, a structure-guided
OKS N222G mutant dramatically extended the product chain length to yield
four novel polyketides including C22 aromatic octaketides from the C6-C2
phenylacetyl starter, as well as a novel C19 heptaketide benzophenone from
the C6-C1 benzoyl starter.
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Abe,
I., Oguro, S., Utsumi, Y., Sano, Y., Noguchi, H., 2005. Engineered
biosynthesis of plant polyketides: chain length control in an
octaketide-producing plant type III polyketide synthase. Journal of the
American Chemical Society 127, 12709-12716.
The chalcone synthase (CHS) superfamily of type III polyketide synthases (PKSs)
produces a variety of plant secondary metabolites with remarkable structural
diversity and biological activities (e.g., chalcones, stilbenes,
benzophenones, acrydones, phloroglucinols, resorcinols, pyrones, and
chromones). Here we describe an octaketide-producing novel plant-specific
type III PKS from aloe (Aloe arborescens) sharing 50-60% amino acid
sequence identity with other plant CHS-superfamily enzymes. A recombinant
enzyme expressed in Escherichia coli catalyzed seven successive
decarboxylative condensations of malonyl-CoA to yield aromatic octaketides
SEK4 and SEK4b, the longest polyketides known to be synthesized by the
structurally simple type III PKS. Surprisingly, site-directed mutagenesis
revealed that a single residue Gly207 (corresponding to the CHS's active
site Thr197) determines the polyketide chain length and product specificity.
Small-to-large substitutions (G207A, G207T, G207M, G207L, G207F, and G207W)
resulted in loss of the octaketide-forming activity and concomitant
formation of shorter chain length polyketides (from triketide to heptaketide)
including a pentaketide chromone, 2,7-dihydroxy-5-methylchromone, and a
hexaketide pyrone, 6-(2,4-dihydroxy-6-methylphenyl)-4-hydroxy-2-pyrone,
depending on the size of the side chain. Notably, the functional diversity
of the type III PKS was shown to evolve from simple steric modulation of the
chemically inert single residue lining the active-site cavity accompanied by
conservation of the Cys-His-Asn catalytic triad. This provided novel
strategies for the engineered biosynthesis of pharmaceutically important
plant polyketides.
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Abe, I., Watanabe, T., Morita, H.,
Kohno, T., Noguchi, H., 2006. Engineered biosynthesis of plant polyketides:
manipulation of chalcone synthase. Organic Letters 8, 499-502.
Chalcone synthase (CHS) is a plant-specific type III polyketide synthase
catalyzing condensation of 4-coumaroyl-CoA with three molecules malonyl-CoA.
Surprisingly, it was demonstrated that S338V mutant of Scutellaria
baicalensis CHS produced octaketides SEK4/SEK4b from eight molecules of
malonyl-CoA. Further, the octaketides-forming activity was dramatically
increased in a CHS triple mutant (T197G/G256L/S338T). The functional
conversion is based on the simple steric modulation of a chemically inert
residue lining the active-site cavity.
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Abe,
I., 2006. Engineered biosynthesis of plant polyketides. Chain length control
in novel type III polyketide synthases. In: ACS Symposium Series #955,
Polyketide: Synthesis, Biological Activity, and Genetic Engineering, American
Chemical Society, pp. 109-127.
A
growing number of functionally divergent type III polyketide synthases (PKSs),
the chalcone synthase (CHS) superfamily enzymes, have been cloned and
characterized, which include recently obtained pentaketide chromone synthase
(PCS) and octaketide synthase (OKS) from aloe (Aloe arborescens).
Recombinant PCS expressed in Escherichia coli catalyzed successive
condensations of malonyl-CoA to produce a pentaketide,
5,7-dihydroxy-2-methylchromone, while recombinant OKS yielded octaketides,
SEK4 and SEK4b, the longest polyketides produced by the structurally simple
type III PKS. PCS and OKS share 92% amino acid sequence identity, and
maintain the conserved Cys-His-Asn catalytic triad. The most characteristic
feature is that the CHS active site residue 197 (numbering in Medicago
sativa CHS) is uniquely replaced with Met in PCS and Gly in OKS,
respectively. Site-directed mutagenesis revealed that the chemically inert
single residue 197 lining the active-site cavity determines the polyketide
chain length and the product specificity depending on the size of the side
chain.
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Carreras, C. W., Pieper, R., Khosla, C., 1996. Efficient synthesis of
aromatic polyketides in vitro by the actinorhodin polyketide synthase.
Journal of the American Chemical Society 118, 5158-5159
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Morita, H., Kondo, S., Kato, R., Wanibuchi, K.,
Noguchi, H., Sugio, S., Abe, I., Kohno, T., 2007.
Crystallization and
preliminary crystallographic analysis of an octaketide-producing plant type
III polyketide synthase.
Acta Crystallograph. Sect. F. Struct. Biol. Cryst. Commun. F63, 947-949.
Octaketide synthase (OKS) from Aloe arborescens is a plant-specific
type III polyketide synthase that produces SEK4 and SEK4b from eight
molecules of malonyl-CoA. Recombinant OKS expressed in Escherichia coli was
crystallized by the hanging-drop vapour-diffusion method. The crystals
belonged to space group I422, with unit-cell parameters a = b = 110.2, c =
281.4 A ¢ª , alpha = beta = gamma = 90.0. Diffraction data were collected to
2.6 A ¢ª resolution using synchrotron radiation at BL24XU of SPring-8.
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