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(Last
modification: 01. June 2009)
Sechs
Kondensationsreaktionen: Aloeson-Synthase (ALS)
(Abe
et al., 2004; Abe et al., 2006;
Mizuuchi et al., 2009)
Diese neuartige Typ III PKS war die erste Pflanzen-PKS, welche mehr als drei
Kondensationen durchführte; die cDNA wurde aus einem Rhabarber kloniert (Rheum
palmatum) und später aus
Aloe arborescens
(Tintenfisch-Aloe). Das rekombinante Protein (genannt
Aloeson-Synthase, ALS),
akzeptierte Acetyl-CoA als Starter, führte sechs Kondensationen mit Malonyl-CoA
durch und eine Zyklisierung zu einem aromatischen Heptaketid (Aloesone, s.
Schema). Das Protein war 60% identisch mit typischen Chalconsynthasen (CHS),
enthielt eine fast identische CoA-Bindungsstelle, und natürlich die katalytisch
wichtigen Aminosäuren, die in allen Proteinen dieser Familie konserviert sind.
Homologie-basierte Modelle sagen in der Tat voraus, dass die generelle Faltung
des Proteins genauso so ist wie bei CHS.
Die zweite Publikation (Abe
et al., 2006) beschrieb eine grosse Zahl an Mutanten, zur Untersuchung der
Frage, welche Aminosäuren wichtige Rollen bei den funktionellen Unterschieden
zwischen ALS und den anderen Typ III PKS spielen. Hier wird nur ein Beispiel
gegeben, weil es zu einem interessanten Ergebnis führte: Die Umwandlung der
Heptaketidsynthase ALS in eine Pentaketidsynthase, vergleichbar mit der
Pentaketidsynthase PCS, die auch auf unserer Website beschrieben wird
(siehe: Chromon-Synthase).
Diese Umwandlung geschah durch die Änderung von Ala197 zu einem Thr, aber sie
war nicht perfekt: Es wurde zwar ein Chromon gebildet, aber nicht das von der
PCS synthetisierte
5,7-Dihydroxy-2-Methylchromon,
sondern das isomere 2,7-Dihydroxy-5-Methylchromon.
Interessanterweise ist dies das gleiche Produkt, welches von einer
Oktaketid-Synthase (OKS)
Mutante gebildet wurde, bei der die Aminosäure an der entsprechenden Position
(Gly) in ein Met umgewandelt wurde: Siehe die
funktionelle Interkonversion von OKS und PCS !
Update
March 13,
2009: A new publication (Mizuuchi
et al., 2009) describes three new type III PKS from Aloe arborescens:
-
PKS4 and PKS5
(ACR19997,
ACR19998)
are functionally identical with the previously described octaketide synthase
(OKS): more...
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PKS3
(ABS72373) turned out to be a multifunctional enzyme: it is a heptaketide
synthase that presumably is involved in the biosynthesis of
aloesone,
i.e. it can be described as
aloesone synthase (ALS) because that was the major product of the
reaction. It also synthesized in smaller amounts two more heptaketides, a
hexaketide (possibly a precursor of aloein), and the octaketides
SEK4/SEK4b which are the in vitro products of the previously
described OKS: more...
-
Mutagenesis
experiments with PKS3:
Interestingly, PKS3 contained an Alanine (A207) in a position where
most other type III PKS like chalcone synthases (CHS) and stilbene synthases
contain a highly conserved Threonine (T197). The enzymes with OKS activity
from the same plant (OKS, PKS4, PKS5) contained a glycine (G207) in this
position, while the pentaketide synthase (PCS) from
Aloe arborescens contained a
methionine. Mutagenesis experiments with A207 in PKS3 then showed:
- A207 mutagenesis to glycine produced an OKS,
- A207 mutagenesis to methionine produced a pentaketide product (Pentaketide
Chromone Synthase, PCS function:
more...).
This is another amazing example
stressing the importance of the amino acid in this particular position for
the capacity to carry out specific numbers of condensation reactions,
leading widely different sizes of the products.
-> Comparable experiments
converting OKS into PCS and vice versa had been carried out before:
more...
Zum Seitenanfang
Links zu den
einzelnen Seiten: Enzyme mit mehr als drei Kondensationen
Zum Seitenanfang
.
Zitate
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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.
Zurück zum Text
-
Abe,
I., Utsumi, Y., Oguro, S., Noguchi, H., 2004. The first plant type III
polyketide synthase that catalyzes formation of aromatic heptaketide. FEBS
Letters 562, 171-176.
A
cDNA encoding a novel plant type III polyketide synthase (PKS) was cloned
from rhubarb (Rheum palmatum). A recombinant enzyme expressed in
Escherichia coli accepted acetyl-CoA as a starter, carried out six
successive condensations with malonyl-CoA and subsequent cyclization to
yield an aromatic heptaketide, aloesone. The enzyme shares 60% amino
acid sequence identity with chalcone synthases (CHSs), and maintains almost
identical CoA binding site and catalytic residues conserved in the CHS
superfamily enzymes. Further, homology modeling predicted that the 43-kDa
protein has the same overall fold as CHS. This provides new insights into
the catalytic functions of type III PKSs, and suggests further involvement
in the biosynthesis of plant polyketides.
Zurück zum Text
-
Abe,
I., Watanabe, T., Lou, W., Noguchi, H., 2006. Active site residues governing
substrate selectivity and polyketide chain length in aloesone synthase. FEBS
Journal 273, 208-218.
Aloesone synthase (ALS) and chalcone synthase (CHS) are plant-specific type
III poyketide synthases sharing 62% amino acid sequence identity. ALS
selects acetyl-CoA as a starter and carries out six successive condensations
with malonyl-CoA to produce a heptaketide aloesone, whereas CHS catalyses
condensations of 4-coumaroyl-CoA with three malonyl-CoAs to generate
chalcone. In ALS, CHS's Thr197, Gly256, and Ser338, the active site residues
lining the initiation elongation cavity, are uniquely replaced with Ala,
Leu, and Thr, respectively. A homology model predicted that the active site
architecture of ALS combines a 'horizontally restricting' G256L substitution
with a 'downward expanding' T197A replacement relative to CHS. Moreover, ALS
has an additional buried pocket that extends into the 'floor' of the active
site cavity. The steric modulation thus facilitates ALS to utilize the
smaller acetyl-CoA starter while providing adequate volume for the
additional polyketide chain extensions. In fact, it was demonstrated that
CHS-like point mutations at these positions (A197T, L256G, and T338S)
completely abolished the heptaketide producing activity. Instead, A197T
mutant yielded a pentaketide, 2,7-dihydroxy-5-methylchromone, while L256G
and T338S just afforded a triketide, triacetic acid lactone. In contrast,
L256G accepted 4-coumaroyl-CoA as starter to efficiently produce a
tetraketide, 4-coumaroyltriacetic acid lactone. These results suggested that
Gly256 determines starter substrate selectivity, while Thr197 located at the
entrance of the buried pocket controls polyketide chain length. Finally,
Ser338 in proximity of the catalytic Cys164 guides the linear polyketide
intermediate to extend into the pocket, thus leading to formation of the
hepataketide in Rheum palmatum ALS.
Zurück zum Text
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