Enzymatic formation of quinolone alkaloids by a plant type III polyketide synthase

Article abstract of DOI:10.1021/ol0625233

(Chemical Equation Presented) Benzalacetone synthase from Rheum palmatum efficiently catalyzed condensation of N-methylanthraniloyl-CoA (or anthraniloyl-CoA) with malonyl-CoA (or methylmalonyl-CoA) to produce 4-hydroxy-2(1H)-quinolones, a novel alkaloidal scaffold produced by a type III polyketide synthase (PKS). Manipulation of the functionally divergent type III PKSs by a nonphysiological substrate thus provides an efficient method for production of pharmaceutically important quinolone alkaloids.

Full text of DOI:10.1021/ol0625233

ORGANIC
LETTERS
2006
Vol. 8, No. 26
6063-6065
Enzymatic Formation of Quinolone
Alkaloids by a Plant Type III Polyketide
Synthase
Ikuro Abe,*,†,‡ Tsuyoshi Abe,† Kiyofumi Wanibuchi,† and Hiroshi Noguchi†
School of Pharmaceutical Sciences and the COE 21 Program, UniVersity of Shizuoka,
Shizuoka 422-8526, Japan, and PRESTO, Japan Science and Technology Agency,
Kawaguchi, Saitama 332-0012, Japan
abei@ys7.u-shizuoka-ken.ac.jp
Received October 13, 2006
ABSTRACT
Benzalacetone synthase from Rheum palmatum efficiently catalyzed condensation of N-methylanthraniloyl-CoA (or anthraniloyl-CoA) with
malonyl-CoA (or methylmalonyl-CoA) to produce 4-hydroxy-2(1H)-quinolones, a novel alkaloidal scaffold produced by a type III polyketide
synthase (PKS). Manipulation of the functionally divergent type III PKSs by a nonphysiological substrate thus provides an efficient method for
production of pharmaceutically important quinolone alkaloids.
The functional diversity and catalytic potential of the
chalcone synthase (CHS) (EC 2.3.1. 74) superfamily of type
III polyketide synthases (PKSs) are remarkable.1 The struc-
turally simple homodimeric proteins catalyze iterative de-
carboxylative condensations of malonyl-CoA with a CoA-
linked starter molecule to produce a variety of biologically
active secondary metabolites. For example, CHS, a pivotal
enzyme in flavonoid biosynthesis, catalyzes sequential
condensation of 4-coumaroyl-CoA with three C2 units from
malonyl-CoA to produce a tetraketide naringenin chalcone
(Scheme 1A),2 whereas benzalacetone synthase (BAS) from
Rheum palmatum (Polygonaceae) carries out a one-step
decarboxylative condensation of 4-coumaroyl-CoA with
malonyl-CoA to produce the C6-C4 skeleton of a diketide
benzalacetone (Scheme 1B).3 One of the most characteristic
features is that plant type III PKSs exhibit unusually broad,
promiscuous substrate specificities; the enzymes readily
accept a variety of nonphysiological substrates, including
aromatic and aliphatic CoA thioesters, to produce a vast array
of chemically and structurally distinct unnatural polyketides.4,5
We now report that the diketide-producing R. palmatum BAS
efficiently catalyzes condensation of N-methylanthraniloyl-
CoA (or anthraniloyl-CoA) with malonyl-CoA (or methyl-
malonyl-CoA) to produce 4-hydroxy-2(1H)-quinolones, a
(2) (a) Ferrer, J. L.; Jez, J. M.; Bowman, M. E.; Dixon, R. A.; Noel, J.
P. Nat. Struct. Biol. 1999, 6, 775-784. (b) Jez, J. M.; Ferrer, J. L.; Bowman,
M. E.; Dixon, R. A.; Noel, J. P. Biochemistry 2000, 39, 890-902. (c) Jez,
J. M.; Noel, J. P. J. Biol. Chem. 2000, 275, 39640-39646. (d) Jez, J. M.;
Bowman, M. E.; Noel, J. P. Biochemistry 2001, 40, 14829-14838. (e) Tropf,
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2000, 122, 11242-11243. (b) Morita, H.; Takahashi, Y.; Noguchi, H.; Abe,
I. Biochem. Biophys. Res. Commun. 2000, 279, 190-195. (c) Morita, H.;
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† University of Shizuoka.
‡ PRESTO.
(1) For recent reviews, see: (a) Schro¨der, J. In ComprehensiVe Natural
Products Chemistry; Elsevier: Oxford, 1999; Vol. 2, pp 749-771. (b)
Austin, M. B.; Noel, J. P. Nat. Prod. Rep. 2003, 20, 79-110.
10.1021/ol0625233 CCC: $33.50
© 2006 American Chemical Society
Published on Web 11/21/2006

Scheme 1. Formation of Polyketides and Acridone/Quinolone Alkaloids by Plant Type III PKSsa
a Yield (%) of quinolone production under the standard assay conditions (see Supporting Information).
novel alkaloidal scaffold produced by a CHS superfamily
type III PKS (Scheme 1D). The quinolone alkaloids6 have
been investigated as N-methyl-D-aspartate7 (NMDA) and
produce 1,3-dihydroxy-N-methylacridone (a tetraketide)
(Scheme 1C);11 however, the enzyme involved in the
biosynthesis of quinolone alkaloids has not been identified
yet. Interestingly, the chalcone-forming CHS and other type
III PKSs, except ACS, do not accept the shorter and the
bulkier N-methylanthraniloyl-CoA as a starter unit despite
their promiscuous substrate specificity. Although the Medi-
cago satiVa CHS F215S mutant has been shown to accept
N-methylanthraniloyl-CoA to produce an unnatural novel
tetraketide lactone after three condensations with malonyl-
CoA,5c enzymatic formation of a quinolone alkaloid (a
diketide) by type III PKSs including mutants of R. graVeolens
ACS11 has not been reported so far.
8
serotonin 5-HT3 receptor antagonists. Moreover, they are
also important intermediates in the chemical synthesis of
alkaloids.9
Anthranilic acid has been postulated to be a key inter-
mediate in the biosynthesis of quinolone and acridone
alkaloids, which occur in the greatest abundance in plants
from the family of Rutaceae (Figure 1).10 In fact, acridone
synthase (ACS) form Ruta graVeolens is a plant-specific type
III PKS that selects N-methylanthraniloyl-CoA as a starter
and performs three condensations with malonyl-CoA to
R. palmatum BAS is the enzyme that catalyzes one-step
condensation with malonyl-CoA to produce a diketide
benzalacetone.3 When recombinant R. palmatum BAS func-
tionally expressed in Escherichia coli was incubated with
(5) (a) Schu¨z, R.; Heller, W.; Hahlbrock, K. J. Biol. Chem. 1983, 258,
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(6) Two-step synthesis of 4-hydroxyquinolinones from anthranilate
derivatives by a tellurium-triggered cyclization reaction has been recently
reported; see: Dittmer, D. C.; Li, Q.; Avilov, D. V. J. Org. Chem. 2005,
70, 4682-4686.
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I. Z. Naturforsch. B: Anorg. Chem., Org. Chem. 1981, 36B, 1176-1179.
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(11) (a) Junghanns, K. T.; Kneusel, R. E.; Baumert, A.; Mainer, W.;
Groger, D.; Matern, U. Plant Mol. Biol. 1995, 27, 681-692. (b) Lukacin,
R.; Springob, K.; Urbanke, C.; Ernwein, C.; Schro¨der, G.; Schro¨der, J.;
Matern, U. FEBS Lett. 1999, 448, 135-140. (c) Springob, K.; Lukacin,
R.; Ernwein, C.; Groning, I.; Matern, U. Eur. J. Biochem. 2000, 267, 6552-
6559. (d) Lukacin, R.; Schreiner, S.; Matern, U. FEBS Lett. 2001, 508,
413-417. (e) Lukacin, R.; Schreiner, S.; Silber, K.; Matern, U. Phytochem-
istry 2005, 66, 277-284.
(7) (a) Rowley, M.; Kulagowski, J. J.; Watt, A. P.; Rathbone, D.;
Stevenson, G. I.; Carling, R. W.; Baker, R.; Marshall, G. R.; Kemp, J. A.;
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Tricklebank, M. D.; Leeson, P. D. J. Med. Chem. 1997, 40, 4053-4068.
(b) Zhou, Z.-L.; Navratil, J. M.; Cai, S. X.; Whittemore, E. R.; Espitia, S.
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6064
Org. Lett., Vol. 8, No. 26, 2006

for the formation of benzalacetone was reported to be at
maximum within a range of pH 8.0-8.5. It should be noted
that R. palmatum BAS exhibited a dramatic change from
benzalacetone to triketide pyrone production at acidic pH.3b
For the quinolone production, the best yield (86% under
the standard assay conditions) was obtained with the
combination of N-methylanthraniloyl-CoA and methyl-
malonyl-CoA. Notably, both esters are nonphysiological
substrates for R. palmatum BAS. On the other hand, a
combination of anthraniloyl-CoA and malonyl-CoA afforded
only 4% yield. Steady-state enzyme kinetics for the formation
of 4-hydroxy-1,3-dimethyl-2(1H)-quinolone (4) revealed KM
) 23.7 µM and kcat ) 1.48 min-1 for N-methylanthraniloyl-
CoA, which was comparable with those for the formation
of benzalacetone, the normal product of the enzyme; KM
)
10.0 µM and kcat ) 1.79 min-1 for 4-coumaroyl-CoA. This
strongly suggests the presence of a not yet identified novel
type III PKS that produces quinolone alkaloids12 from the
CoA thioester of anthranilic acid (Figure 1).
In R. graVeolens ACS, the residues Ser132, Ala133, and
Val265 (numbering in M. satiVa CHS) have been proposed
to play a critical role for the selection of N-methylanthra-
niloyl-CoA as a starter substrate and for the formation of
the acridone alkaloid.11d Indeed, an ACS triple mutant
(S132T/A133S/V265F) has been shown to yield an enzyme
that was functionally identical with CHS.11d However,
interestingly, two of the residues are not conserved in R.
palmatum BAS (S132L/V265F) that accepted the anthra-
niloyl starter. On the other hand, the diketide-forming activity
of the BAS enzyme is derived from the characteristic
substitution of the active-site gatekeeper Phe215; the con-
formationally flexible residue conserved in all the known
type III PKSs is uniquely replaced with nonaromatic Leu in
BAS.3 Although the BAS L215F mutant has been shown to
recover chalcone-forming activity to produce chalcone after
three condensations with malonyl-CoA,3b the mutant did not
produce the tetraketide acridone from the anthraniloyl starter
but instead just yielded the diketide quinolone. These results
suggest subtle differences of the active-site structure between
R. palmatum BAS and R. graVeolens ACS.
Figure 1. Naturally occurring quinoline alkaloids derived from
4-hydroxy-2(1H)-quinolones from Rutaceae plants.12
N-methylanthraniloyl-CoA (or anthraniloyl-CoA) and ma-
lonyl-CoA (or methylmalonyl-CoA) as substrates, the en-
zyme efficiently afforded 4-hydroxy-2(1H)-quinolones (1-
4) as a single product (Scheme 1D). The condensation
products were not detected in control experiments performed
with a boiled enzyme preparation. The spectroscopic data
1
(LC-ESIMS, UV, and H NMR) of the compounds were
characteristic of those of the quinolone alkaloids and
completely identical with those of authentic compounds.6 For
example, the LC-ESIMS spectrum of 4-hydroxy-1-methyl-
2(1H)-quinolone (3) gave a parent ion peak [M + H]+ at
m/z 176, indicating the structure of a diketide. The 1H NMR
of 3 obtained from a large-scale enzyme reaction (0.4 mg
from 2 mg of N-methylanthraniloyl-CoA and 4 mg of
malonyl-CoA) showed one methyl singlet (δ 3.52) in addition
to one vinyl proton (δ 5.86) and four aromatic protons (δ
7.89-7.17), supporting that the heterocyclic system adopts
the more stable 4-hydroxy-2(1H)-quinolone form. Confirma-
tion of the structure was finally obtained by direct compari-
son with the commercially available authentic compound.
These quinolone alkaloids have never been isolated from R.
palmatum (Polygonaceae).
In summary, the present work describes the first enzymatic
synthesis of 4-hydroxy-2(1H)-quinolones. Manipulation of
the functionally divergent CHS superfamily type III PKSs
by nonphysiological substrates thus provides an efficient
method for production of pharmaceutically important plant
alkaloids.
Acknowledgment. This work was supported by the
PRESTO program from the Japan Science and Technology
Agency, a Grant-in-Aid for Scientific Research (Nos.
18510190 and 17310130), and the COE21 program from the
Ministry of Education, Culture, Sports, Science and Technol-
ogy, Japan.
Interestingly, the enzyme reaction with the anthraniloyl-
CoA proceeds without the “decarboxylation” step, and the
amide formation immediately follows the condensation
reactions of N-methylanthraniloyl-CoA (or anthraniloyl-CoA)
and malonyl-CoA (or methylmalonyl-CoA) (Scheme 1D).
This was also the case for the conversion of benzoyl-CoA
(i.e., without the methylamino group of N-methylanthra-
niloyl-CoA) to 6-phenyl-4-hydroxy-2-pyrone (a triketide) by
R. palmatum BAS.3b Furthermore, the quinolone formation
activity showed a broad pH optimum within a range of pH
6.0-8.5. Thus, the pH change did not affect the product
profile; formation of triketides or other polyketides was not
detected in the reaction mixture. In contrast, the pH optimum
Supporting Information Available: Materials and meth-
ods. This material is available free of charge via the Internet
at http://pubs.acs.org.
OL0625233
(12) Michael, J. P. Nat. Prod. Rep. 2004, 21, 650-668.
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