(Last
modification:
12. May 2010) Eine Kondensation: Benzalaceton
Rhabarber (Rheum palmatum) Himbeeren sind nicht die einzigen Pflanzen mit Benzalaceton-Derivaten, und tatsächlich wurde die erste BAS aus einer anderen Pflanze kloniert: Einer Rhabarber-Art (Rheum palmatum ) (Abe et al., 2001). Wissen Sie, wie diese Pflanze aussieht? Hier klicken.
Danach wurden mit diesem Enzym eine Reihe von interessanten Mutagenese-Studien durchgeführt, welche die Frage untersuchten, was denn eine BAS-Funktionalität ausmacht. Ein interessantes Ergebnis wurde erst bei der vergleichenden Analyse der Mutanten erhalten. Unter den Standard-Testbedingungen, d.h. bei pH 8-8.5, führte das unveränderte Enzym wirklich nur eine einzige Kondensation durch. Wurde der pH jedoch auf 6.0 abgesenkt, zeigte sich eine dramatische Veränderung: Anstatt des Benzalacetons synthetisierte das Enzym jetzt Bisnoryangonin, d.h. es führte zwei Kondensationen statt einer durch, und das praktisch zu 100% ! Unter diesen Bedingungen in vitro war das Protein also eine Bisnoryangonin-Synthase, nicht eine BAS (s.
auch weiter unten). Das Schema zeigt die Reaktionen. Nach meinem Wissen ist unbekannt, ob dies in der Pflanze eine Rolle spielt. Im Zweifelsfalle ist dies wieder ein Fall, dass Reaktionen in vitro nicht sonderlich aussagekräftig sind:
Mehr...
pH-Abhängigkeit der Benzalaceton-Synthase (BAS)
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Important update February 2010
The crystal structure of this BAS and
with it the detailed analysis of the active site pocket and the reaction have
been published: Morita et
al., 2010.
See also: Abe et al., 2007
and Morita
et al., 2008.
A
brief summary oft he most important points (you really should look in detail at
the publication: there is much more of interest in it!).
Note: For convenience, I will use the numbering of the CHS from
Medicago sativa throughout this brief discussion.
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Overall structure
It is really very similar to all other crystallized type III PKS, the
interesting details are in the active site architecture.
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Active site cavity
As noted before, from models as well as from mutagenesis experiments, one of
the most important features is the replacement of the Leu214/Phe215
conserved in CHS by the Ile/Leu in BAS: the Leu side chain protrudes into
the the active site cavity of BAS, thus restricting its size.
Interesting is the position of the side chain of Ser338: the hydroxyl group
rotates by nearly 120 degrees, and with that blocks the entrance of the
coumaroyl-binding pocket present in CHS. Actually BAS uses an alternative
pocket to lock the coumaroyl-residue. As a result of these two effects, the
total cavity volume in BAS is less than 50% of that in CHS, restricting the
number of condensations.
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Active site entrance
The Phe215/Leu exchange has another effect: it enlarges the active site
entrance of BAS; it is about twice as large as that of CHS. This may well
explain the previous finding that BAS can accept the bulky N-methylanthraniloyl-CoA
as substrate, carrying out a single condensation, and releasing a quinoline
as product: more…. There
are similarities to the previous finding that the substitution of Phe215 in
M. sativa CHS by the much smaller Ser led to a larger opening of the
active site, and thus allowed the use of N-methylanthraniloyl-CoA as
substrate. Interestingly, that mutant could carry out three condensations,
synthesizing N-methylanthraniloyltriacetic acid lactone as
product (Jez et al., 2002).
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Thioester bond cleavage and decarboxylation to the end product
Cleavage of the thioester bond of the enzyme-bound tetraketide intermediate
is an important trigger for the aldol condensation in the stilbene synthase
(STS) type cyclization/decarboxylation, and the mechanism has been called
“aldol switch” (more…). It is
therefore an intriguing question whether the BAS reaction, with release of
the diketide and a decarboxylation, follows the same mechanisms. However,
that seems not possible because one of the residues critical for the aldol
switch, a Thr, is replaced by a Leu in BAS (more…).
The structure suggested instead that there is a hydrogen bond network with
the Cys-His-Asn catalytic triad. The authors therefore propose that an
enolate anion of the diketide is stabilized by the His-Asn oxyanion hole,
and that this promotes the decarboxylation of the diketide to the
benzalacetone, with the same mechanism as in the decarboxylation of the
malonyl-CoA (Ferrer et al.,
1999).
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Restoration of CHS activity
Changing the Ile/Leu in BAS to the Leu214/Phe215 conserved in CHS had some
interesting effects. One is that it apparently restored the coumaroyl-binding
pocket present in CHS; with the result that the mutant at least partially
now had the catalytic properties of CHS. To evaluate the significance, one
has to look at the pH-dependencies (also see above the properties of the
wild-type BAS). The mutant had an increased tendency to carry out more than
one condensation: benzalacetone was a minor product (still best at high pH),
but the most abundant product was now bisnoryangonin (two condensations),
with a pH optimum shifted from 6-6.5 (parent) to pH 7-8.5 (mutant).
Products from three condensations were also detected, but at lower amounts
than bisnoryangonin: CTAL (three condensations, but no cyclization to
chalcone) and indeed a bit of naringenin chalcone. In both cases the pH
optimum was slightly acidic (pH 6.5), and hardly any of these products were
detectable at pH 8 (Abe et al., 2003),
and Figure below.
pH dependence of
enzyme activities of wild-type BAS and the mutant to CHS (Ile/Leu
in BAS to the Leu214/Phe215 conserved in CHS).
BNY, Bisnoryangonin; CTAL,
4-Coumaroyltriacetic acid lactone.
This is part of Fig. 6 (rearranged) in Morita et al. (2010)
All in all, these are
very interesting and important findings, with identification of structural
elements important for the enzyme reaction.
There is just one small point that I think could have been discussed a
bit more: what could be the molecular basis of the pronounced pH-dependence?
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Seitenanfang
Zitate -
Morita,
H., Shimokawa, Y., Tanio, M., Kato, R., Noguchi, H., Sugio, S., Kohno, T.,
Abe, I., 2010. A structure-based mechanism for benzalacetone synthase from
Rheum palmatum. Proceedings of the National Academy of Sciences of
the United States of America 107, 669-673.
Benzalacetone synthase (BAS), a plant-specific type III polyketide synthase
(PKS), catalyzes a one-step decarboxylative condensation of malonyl-CoA and
4-coumaroyl-CoA to produce the diketide benzalacetone. We solved the crystal
structures of both the wild-type and chalcone-producing I207L/L208F mutant
of Rheum palmatum BAS at 1.8 A resolution. In addition, we solved the
crystal structure of the wild-type enzyme, in which a monoketide coumarate
intermediate is covalently bound to the catalytic cysteine residue, at 1.6 A
resolution. This is the first direct evidence that type III PKS utilizes the
cysteine as the nucleophile and as the attachment site for the polyketide
intermediate. The crystal structures revealed that BAS utilizes an
alternative, novel active-site pocket for locking the aromatic moiety of the
coumarate, instead of the chalcone synthase's coumaroyl-binding pocket,
which is lost in the active-site of the wild-type enzyme and restored in the
I207L/L208F mutant. Furthermore, the crystal structures indicated the
presence of a putative nucleophilic water molecule which forms hydrogen bond
networks with the Cys-His-Asn catalytic triad. This suggested that BAS
employs novel catalytic machinery for the thioester bond cleavage of the
enzyme-bound diketide intermediate and the final decarboxylation reaction to
produce benzalacetone. These findings provided a structural basis for the
functional diversity of the type III PKS enzymes.
Zurück
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Abe, T., Morita, H., Noma, H., Kohno,
T., Noguchi, H., Abe, I., 2007.
Structure function analysis of benzalacetone synthase from Rheum palmatum.
Bioorganic & Medicinal Chemistry Letters 17, 3161-3166.
Benzalacetone synthase (BAS) is a plant-specific chalcone synthase
(CHS) superfamily type III polyketide synthase (PKS) that catalyzes a
one-step decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA.
The diketide forming activity of Rheum palmatum BAS is attributed to
the characteristic substitution of the conserved active-site Phe215 with Leu
(numbering in Medicago sativa CHS). To further understand the
structure and function of R. palmatum BAS, four site-directed mutants
(C197T, C197G, G256L, and S338V) were newly constructed. All the mutants did
not change the product pattern, however, the activity was 2-fold increased
in S338V, while reduced to half in G256L mutant. On the other hand, the C197
mutants were functionally almost identical to wild-type BAS, excluding the
possibility that the second active-site Cys is involved in the enzyme
reaction. Instead, homology modeling suggested a possibility that, unlike
the case of CHS, BAS utilizes an alternative pocket to lock the coumaroyl
moiety for the diketide formation reaction.
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Morita, H., Tanio, M., Kondo, S., Kato, R.,
Wanibuchi, K., Noguchi, H., Sugio, S., Abe, I., Kohno, T., 2008.
Crystallization and preliminary crystallographic analysis of a plant type
III polyketide synthase that produces benzalacetone. Acta Crystallographica
Section F: Structural Biology and Crystallization Communications 64,
304-306.
Benzalacetone synthase (BAS) from Rheum palmatum is a
plant-specific type III polyketide synthase that catalyzes the one-step
decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce
the diketide 4-(4-hydroxyphenyl)-but-3-en-2-one. Recombinant BAS expressed
in Escherichia coli was crystallized by the sitting-drop
vapour-diffusion method. The crystals belong to space group P2(1), with
unit-cell parameters a = 54.6, b = 89.6, c = 81.1 A, alpha = gamma = 90.0,
beta = 100.5 degrees . Diffraction data were collected to 1.8 A resolution
using synchrotron radiation at BL24XU of SPring-8.
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Jez, J. M., Bowman, M. E.,
Noel, J. P., 2002. Expanding the biosynthetic repertoire of plant type
III polyketide synthases by altering starter molecule specificity. Proceedings
of the National Academy of Sciences of the United States of America 99,
5319-5324.
Type III polyketide synthases (PKS) generate an array of natural products by
condensing multiple acetyl units derived from malonyl-CoA to
thioester-linked starter molecules covalently bound in the PKS active
site. One strategy adopted by Nature for increasing the functional
diversity of these biosynthetic enzymes involves modifying polyketide
assembly by altering the preference for starter molecules. Chalcone
synthase (CHS) is a ubiquitous plant PKS and the first type III PKS
described functionally and structurally. Guided by the
three-dimensional structure of CHS, Phe-215 and Phe-265, which are
situated at the active site entrance, were targeted for site-directed
mutagenesis to diversify CHS activity. The resulting mutants were
screened against a panel of aliphatic and aromatic CoA-linked starter
molecules to evaluate the degree of starter molecule specificity in
CHS. Although wild-type CHS accepts a number of natural CoA
thioesters, it does not use N-methylanthraniloyl-CoA as a
substrate. Substitution of Phe-215 by serine yields a CHS mutant that
preferentially accepts this CoA-thioester substrate to generate a
novel alkaloid, namely N-methylanthraniloyltriacetic acid
lactone. These results demonstrate that a point mutation in CHS
dramatically shifts the molecular selectivity of this enzyme. This
structure-based approach to metabolic redesign represents an initial
step toward tailoring the biosynthetic activity of plant type III PKS.
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Abe, I., Takahashi, Y., Morita, H. and Noguchi, H., 2001. Benzalacetone synthase: a novel polyketide synthase that plays a crucial role in the biosynthesis of phenylbutanones in Rheum palmatum. European Journal of Biochemistry 268, 3354-3359.
Benzalacetone
synthase (BAS) is a novel plant-specific polyketide synthase that catalyzes
a one step decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA
to produce the C 6 -C 4 skeleton of phenylbutanoids in higher plants. A cDNA
encoding BAS was for the first time cloned and sequenced from rhubarb (Rheum
palmatum), a medicinal plant rich in phenylbutanoids including
pharmaceutically important phenylbutanone glucoside, lindleyin (anti-inflammatory
action in extracts, Fig. 4: derivative of raspberry ketone, by attaching a
sugar+phenyl-derivative to hydroxy group of coumaroyl starter residue). The
cDNA encoded a 42 kDa protein that shares 60-75% amino acid sequence
identity with other members of the CHS-superfamily enzymes. Interestingly,
R. palmatum BAS lacks the active-site Phe215 residue (numbering in
CHS) which has been proposed to help orient substrates and intermediates
during the sequential condensation of 4-coumaroyl-CoA with malonyl-CoA in
CHS. On the other hand, the catalytic cysteine-histidine dyad (Cys164 -
His303) in CHS is well conserved in BAS. A recombinant enzyme expressed in
E. coli efficiently afforded benzalacetone as a single product from
4-coumaroyl-CoA and malonyl-CoA. Further, in contrast with CHS that showed
broad substrate specificity toward aliphatic CoA esters, BAS did not accept
hexanoyl-CoA, isobutyryl-CoA, isovaleryl-CoA, and acetyl-CoA as a substrate.
Finally, besides the phenylbutanones in rhubarb, BAS has been proposed to
play a crucial role for the construction of the C 6 -C 4 moiety of a variety
of natural products such as medicinally important gingerols in ginger plant.
Zurück zum Text -
Abe,I.; Sano,Y.; Takahashi,Y.; Noguchi,H., 2003. Site-directed mutagenesis of benzalacetone synthase: the role of Phe215 in plant type III polyketide synthases. Journal of Biological Chemistry 278, 25218-25226.
Benzalacetone synthase (BAS) and chalcone synthase (CHS) are plant-specific
type III polyketide synthases (PKSs) that share [~]70% amino acid sequence
identity. BAS catalyzes a one-step decarboxylative condensation of
4-coumaroyl-CoA with malonyl-CoA to produce a diketide benzalacetone,
whereas CHS performs sequential condensations with three malonyl-CoA to
generate a tetraketide chalcone. A homology model suggested that BAS has the
same overall fold as CHS with cavity volume almost as large as that of CHS.
One of the most characteristic features is that Rheum palmatum BAS lacks
active site Phe-215; the residues 214LF conserved in type III PKSs are
uniquely replaced by IL. Our observation that the BAS I214L/L215F mutant
exhibited chalcone-forming activity in a pH-dependent manner supported a
hypothesis that the absence of Phe-215 in BAS accounts for the interruption
of the polyketide chain elongation at the diketide stage. On the other hand,
Phe-215 mutants of Scutellaria baicalensis CHS (L214I/F215L, F215W, F215Y,
F215S, F215A, F215H, and F215C) afforded increased levels of truncated
products; however, none of them generated benzalacetone. These results
confirmed the critical role of Phe-215 in the polyketide formation reactions
and provided structural basis for understanding the structure-function
relationship of the plant type III PKSs.
Zurück zum Text -
Austin, M. B., Noel, J. P., 2003.
The chalcone synthase superfamily of type III polyketide synthases.
Natural Product Reports 20, 79-110.
Covering 1970–2001.
This review covers the functionally diverse type III polyketide synthase (PKS)
superfamily of plant and bacterial biosynthetic enzymes, from the discovery
of chalcone synthase (CHS) in the 1970s through the end of 2001. A broader
perspective is achieved by a comparison of these CHS-like enzymes to
mechanistically and evolutionarily related families of enzymes, including
the type I and type II PKSs, as well as the thiolases and -ketoacyl
synthases of fatty acid metabolism. As CHS is both the most frequently
occurring and best studied type III PKS, this enzyme's structure and
mechanism is examined in detail. The in vivo functions and biological
activities of several classes of plant natural products derived from
chalcones are also discussed. Evolutionary mechanisms of type III PKS
divergence are considered, as are the biological functions and activities of
each of the known and functionally divergent type III PKS enzyme families (currently
twelve in plants and three in bacteria). A major focus of this review is the
integration of information from genetic and biochemical studies with the
unique insights gained from protein X-ray crystallography and homology
modeling. This structural approach has generated a number of new predictions
regarding both the importance and mechanistic role of various amino acid
substitutions observed among functionally diverse type III PKS enzymes.
Zurück zum Text
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Ferrer, J.-L., Jez,
J. M., Bowman, M. E., Dixon, R. A., Noel, J. P., 1999. Structure of
chalcone synthase and the molecular basis of plant polyketide biosynthesis.
Nature Structural Biology 6, 775-784.
Chalcone synthase (CHS) is pivotal for the biosynthesis of flavonoid
antimicrobial phytoalexins and anthocyanin pigments in plants, It produces
chalcone by condensing one p-coumaroyl- and three malonyl-coenzyme A
thioesters into a polyketide reaction intermediate that cyclizes, The
crystal structures of CHS alone and complexed with substrate and product
analogs reveal the active site architecture that defines the sequence and
chemistry of multiple decarboxylation and condensation reactions and
provides a molecular understanding of the cyclization reaction leading to
chalcone synthesis. The structure of CHS complexed with resveratrol also
suggests how stilbene synthase, a related enzyme, uses the same substrates
and an alternate cyclization pathway to form resveratrol, By using the
three-dimensional structure and the large database of CHS-like sequences, we
can identify proteins likely to possess novel substrate and product
specificity. The structure elucidates the chemical basis of plant polyketide
biosynthesis and provides a framework for engineering CHS-like enzymes to
produce new products.
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