Substrate specificity of chalcone synthase: Enzymatic formation of unnatural polyketides from synthetic cinnamoyl-CoA analogues [9]

Full text of DOI:10.1021/ja0027113

11242
J. Am. Chem. Soc. 2000, 122, 11242-11243
Scheme 1. Proposed Mechanism for the Conversion of
p-Coumaroyl-CoA (1a) and Its Analogs (1b-e) to Chalcones
(2a,b), Flavanones (3a,b), BNY-type (4a-e), and CTAL-type
Compounds (5a-e)
Substrate Specificity of Chalcone Synthase:
Enzymatic Formation of Unnatural Polyketides from
Synthetic Cinnamoyl-CoA Analogues
Ikuro Abe,* Hiroyuki Morita, Ayumi Nomura, and
Hiroshi Noguchi
School of Pharmaceutical Sciences
UniVersity of Shizuoka, 52-1 Yada
Shizuoka 422-8526, Japan
ReceiVed July 24, 2000
Chalcone synthase (CHS) (EC 2.3.1.74) is a plant specific
polyketide synthase that plays a pivotal role in the biosynthesis
of flavonoids.1 CHS catalyzes a sequential condensation of the
phenylpropanoid unit of p-coumaroyl-CoA (1a) as a starter with
three two-carbon units from malonyl-CoA (Scheme 1). The
reaction is thought to be initiated by binding of p-coumaroyl-
CoA followed by formation of a coumaroyl thioester at Cys164
at the active site of the enzyme. After three rounds of stepwise
condensation of acetate units from malonyl-CoA, cyclization and
aromatization of the enzyme-bound tetraketide intermediate lead
to formation of naringenin chalcone (2a). Naringenin chalcone
is then converted to (-)-(2S)-5,7,4′-trihydroxyflavanone (narin-
genin) by chalcone isomerase; however, in the absence of the
enzyme, chalcone spontaneously forms racemic (2RS)-naringenin
(3a) through a nonstereospecific ring-C closure (Scheme 1).2
CHSs, along with other plant CHS-like enzymes including
stilbene synthase, have been cloned from more than 40 plant
species, and many of them have been functionally expressed in
E. coli.1 CHS functions as a homodimer of a 42 kDa polypeptide.
The recently reported three-dimensional crystal structure of CHS
from alfalfa (Medicago satiVa) revealed that the dimer contains
two functionally independent active sites: the coumaroyl-binding
pocket and the cyclization pocket, defined by four residues
conserved in all the known CHS-related enzymes (Cys164,
Phe215, His303, and Asn336).3 The coumaroyl-binding pocket
has been proposed to lock the moiety in the position, while the
cyclization pocket accommodates the elongating polyketide and
this is where the cyclization and aromatization of the new ring
takes place.4
In this paper, we report substrate specificity of CHS from
Scutellaria baicalensis,5,6 using chemically synthesized substrate
analogues, and describe enzymatic formation of novel unnatural
polyketides. First, we prepared p-coumaroyl-CoA analogues in
which the 4-hydroxyl group was substituted by halogen or a
methoxy group (1b-e) (Scheme 1) to test the effect of the
substitution on the enzyme reaction. We then tested analogues in
which the coumaroyl aromatic ring was replaced by heteroaro-
matic moieties, furan (1f) or thiophene (1g) (Scheme 2). The two-
step synthesis of the cinnamoyl-CoA analogues (1b-e) involved
generation of the N-hydroxysuccinimide esters of the correspond-
ing 4-substituted cinnamic acid followed by a thioester exchange
with CoA as originally described by Sto¨ckigt and Zenk (see
Supporting Information).7 Analogue compounds 1f and 1g were
prepared in the same way from commercially available trans-3-
furanacrylic acid and trans-3-(3-thienyl)acrylic acid, respectively.
When 4-fluorocinnamoyl-CoA (1b) was incubated with the
recombinant CHS,8 three products were isolated by HPLC. The
first product showed a UV spectrum (λmax 296 nm) similar to
that of naringenin (3a), suggesting the structure of (2RS)-4′-fluoro-
5,7-dihydroxyflavanone (3b) (Scheme 1). The LC-ESIMS spec-
trum gave a parent ion peak [M - H]- at m/z 273, and in MS/
MS (precursor ion at m/z 273), the fragment at m/z 151
corresponded to [M - H - C6H4F - C2H3]-. Furthermore, the
1H NMR spectrum of 3b obtained from a large scale incubation
(20% yield from 10 mg of 1b) showed two aromatic protons of
(6) The deduced amino acid sequences of S. baicalensis CHS showed 77.4%
(302/389) identity with those of the alfalfa (M. satiVa) CHS. The recombinant
enzyme with an additional hexahistidine tag at the C-terminal was expressed
in E. coli, and purified by Ni-chelate chromatography as described before.5
The enzyme showed KM ) 36.1 µM and kcat ) 1.26 min-1 for p-coumaroyl-
CoA.
(7) Sto¨ckigt, J.; Zenk, M. H. Z. Naturforsch. 1975, 30c, 352-358.
(8) The standard reaction mixture contained 27 nmol of cinnamoyl-CoA
analogue, 54 nmol of malonyl-CoA, 105 pmol of purified recombinant CHS,
and 1 mM EDTA in a final volume of 500 µL of 100 mM potassium phosphate
buffer, pH 7.5. Incubations were carried out at 30 °C for 1 h, and stopped by
adding 50 µL of 20% HCl. After extraction with ethyl acetate, reaction products
were analyzed by reverse-phase HPLC and LC-ESIMS. For large-scale
reactions, 4-fluorocinnamoyl-CoA (1b) (10.0 mg, 10.9 µmol), trans-3-
furanacryloyl-CoA (1f) (10.0 mg, 11.3 µmol), or trans-3-(3-thienyl)acryloyl-
CoA (1g) (10.0 mg, 11.1 µmol) were respectively incubated with 56 mg of
purified recombinant CHS in 400 mL of 100 mM phosphate buffer containing
malonyl-CoA (20.0 mg, 23.1 µmol) and 1 mM EDTA at 30 °C for 3 h.
* To whom correspondence should be addressed. Tel/Fax: +81-54-264-
5662. E-mail: abei@ys7.u-shizuoka-ken.ac.jp.
(1) Schro¨der, J. In ComprehensiVe Natural Products Chemistry; Sankawa,
U., Ed.; Elsevier: Oxford, 1999; Vol. 1, pp 749-771.
(2) Hahlbrock, K.; Zilg, H.; Grisebach, H. Eur. J. Biochem. 1970, 15, 13-
18.
(3) Ferrer, J.-L.; Jez, J. M.; Bowman, M. E.; Dixon, R. A.; Noel, J. P.
Nature Struct. Biol. 1999, 6, 775-783.
(4) Jez, J. M.; Ferrer, J.-L.; Bowman, M. E.; Dixon, R. A.; Noel, J. P.
Biochemistry 2000, 39, 890-902.
(5) Morita, H.; Noguchi, H.; Akiyama, T.; Shibuya, M.; Ebizuka, Y. In
Towards Natural Medicine Research in the 21st Century, Excerpt Medica
International Congress Series 1157; Elsevier: Tokyo, 1998; pp 401-409.
10.1021/ja0027113 CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/26/2000

Communications to the Editor
J. Am. Chem. Soc., Vol. 122, No. 45, 2000 11243
Scheme 2. Proposed Mechanism for the Conversion of
p-Coumaroyl-CoA Analogs 1f and 1g
and 5b. In contrast, incubation of other 4-substituted analogues
(1c-e, X ) Cl, Br, and OCH3) did not yield flavanones, but
afforded only the BNY- and CTAL-type products (Scheme 1). It
appears that the steric and/or electronic perturbations by the
substituents (4-Cl, Br, or OCH3) larger in size than that of the
natural substrate (4-OH) may alter the stability of the enzyme-
bound tetraketide intermediate or the optimally folded conforma-
tion in the cyclization pocket of the active site of the enzyme.
Interestingly, CHS accepted analogues 1f and 1g, in which the
coumaroyl ring was replaced by heteroaromatic moieties, furan
or thiophene, leading to formation of novel polyketides 3f and
3g (Scheme 2). From large-scale incubations, 3f and 3g were
obtained in 22% and 28% yield, respectively. The LC-ESIMS
spectrum of 3f gave a parent ion peak [M - H]- at m/z 245, and
in MS/MS (precursor ion at m/z 245), the fragment at m/z 151
1
corresponded to [M - H - C4H3O - C2H3]-. Further, the H
NMR spectrum of 3f showed two aromatic protons of ring-A (δ
5.86 and 5.84) coupled to each other, and two R-carbonyl H-3
protons (δ 3.00 and 2.72) coupled to the H-2 proton of ring-C (δ
5.43), clearly confirming the structure. In a similar manner, the
structure of 3g was determined. In addition, as in the case of
other analogues, incubation of 1f and 1g also afforded the pyrone
byproducts (Scheme 2).
This is the first demonstration of the enzymatic formation of
novel, unnatural polyketides containing the heteroaromatic ring
moiety. In particular, it was noteworthy that the enzyme accepted
the CoA ester with furan or thiophene smaller in size than the
p-coumaroyl moiety, and locked in the coumaroyl-binding pocket,
the enzyme-bound thioester efficiently initiated the subsequent
chain elongation and aromatization reactions. Presumably, the
π-π interaction between the substrate and the active site aromatic
residue (e.g. Phe215)4 would play an important role for guiding
the course of the reaction. On the other hand, CHSs have been
reported to also accept aliphatic CoA esters (e.g. hexanoyl-CoA
and isovaleryl-CoA) as starter molecules to produce phloroa-
cylphenones.11,12 In contrast, as described, 4-substituted cinnnamoyl-
CoA analogues (1c-e) only afforded pyrone derivatives, sug-
gesting that the steric size of the substrate is crucial for the
optimally folded conformation of the polyketide intermediate in
the cyclization pocket.
Finally, since as medicinal natural products phenylpropanoids
such as chalcone and stilbene exhibit cancer chemopreventive,
anti-mitotic, estrogenic, anti-malarial, and anti-asthmatic activi-
ties,3 the observed enzyme reaction products may show a
promising profile of important biological activities. Manipulation
of the biosynthesis of phenylpropanoids by synthetic analogues
would thus lead to development of the chemical library of
pharmaceutically important novel polyketides.
ring-A (δ 6.01 and 5.99) and two R-carbonyl H-3 protons (δ 3.17
and 2.81) coupled to H-2 proton (δ 5.58), confirming the structure.
The other two products respectively showed a UV spectrum
similar to that of bis-noryangonin (BNY) (4a)9 and p-couma-
royltriacetic acid lactone (CTAL) (5a),10 thus suggesting the
structures of 4b and 5b (Scheme 1). The pyrone derivatives, BNY
and CTAL, have been known to be common byproducts of CHS
enzyme reactions in Vitro when the reaction mixtures are acidified
before extraction.10 The LC-ESIMS spectrum of BNY-type
compound 4b gave a parent ion peak [M - H]- at m/z 231,
indicating the reaction had terminated after two condensation
reactions of malonyl-CoA, and in MS/MS (precursor ion at m/z
231), the fragment at m/z 187 corresponded to [M - H - CO2]-,
consistent with the presence of a R-pyrone ring. While the LC-
ESIMS of CTAL-type compound 5b gave a parent ion peak [M
- H]- at m/z 273, indicating the successive condensation of three
units of malonyl-CoA, in MS/MS (precursor ion at m/z 273), the
fragment at m/z 229 corresponded to [M - H - CO2]-, indicating
the presence of a R-pyrone ring. The reaction was therefore
terminated without aromatic ring formation.
Acknowledgment. We are indebted to Mr. Kenji Matsuura (JEOL)
for HR-MS measurements. This work was in part supported by The San-
Ei Gen Foundation for Food Chemical Research Grant to I.A.
Supporting Information Available: Chemical synthesis of substrate
analogues, enzyme reaction conditions, HPLC and LC-ESIMS procedure,
and a complete set of spectroscopic data for all the synthetic and the
enzyme reaction products (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
4-Fluorocinnamoyl-CoA (1b) was thus enzymatically converted
to fluorinated flavanone 3b along with the pyrone byproducts 4b
JA0027113
(9) Kreuzaler, F.; Hahlbrock, K. Arch. Biochem. Biophys. 1975, 169, 84-
90.
(11) Schu¨z, R.; Heller, W.; Hahlbrock, K. J. Biol. Chem. 1983, 258, 6730-
6734.
(10) Akiyama, T.; Shibuya, M.; Liu, H.-M.; Ebizuka, Y. Eur. J. Biochem.
1999, 263, 834-839.
(12) Zuurbier, K. W. M.; Leser, J.; Berger, T.; Hofte, A. J. P.; Schro¨der,
G.; Verpoorte, R.; Schro¨der, J. Phytochemistry 1998, 49, 1945-1951.