Engineered biosynthesis of plant polyketides: Manipulation of chalcone synthase

Article abstract of DOI:10.1021/ol052912h

Chalcone synthase (CHS) is a plant-specific type III polyketide synthase catalyzing condensation of 4-coumaroyl-CoA with three molecules of 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.

Full text of DOI:10.1021/ol052912h

ORGANIC
LETTERS
2
006
Vol. 8, No. 3
99-502
Engineered Biosynthesis of Plant
Polyketides: Manipulation of Chalcone
Synthase
4
Ikuro Abe,*,†,§ Tatsuya Watanabe, Hiroyuki Morita, Toshiyuki Kohno, and
Hiroshi Noguchi
†
‡
‡
†
School of Pharmaceutical Sciences and the COE 21 Program, UniVersity of Shizuoka,
Shizuoka 422-8526, Japan, PRESTO, Japan Science and Technology Agency,
Kawaguchi, Saitama 332-0012, Japan, and Mitsubishi Kagaku Institute of Life
Sciences (MITILS), Machida, Tokyo 194-8511, Japan
abei@ys7.u-shizuoka-ken.ac.jp
Received December 1, 2005
ABSTRACT
Chalcone synthase (CHS) is a plant-specific type III polyketide synthase catalyzing condensation of 4-coumaroyl-CoA with three molecules of
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.
The chalcone synthase (CHS) superfamily of type III
polyketide synthases (PKSs) share a common three-
dimensional overall fold with a conserved Cys-His-Asn
catalytic triad to produce structurally diverse polyphenols
royltriacetic acid lactone (CTAL) as early-released derailment
byproducts (Figure 1A). On the other hand, recently reported
octaketide synthase (OKS) from Aloe arborescens catalyzes
2
condensation of eight molecules of malonyl-CoA to produce
1
3b
with remarkable biological activities. CHS is the well-
a 1:4 mixture of octaketide SEK4 and SEK4b (Figure 1B),
characterized plant-specific type III PKS that produces
naringenin chalcone (4,2′,4′,6′-tetrahydroxychalcone), the
biosynthetic precursor of flavonoids, through a sequential
condensation of 4-coumaroyl-CoA with three molecules of
malonyl-CoA (Figure 1A). In an in Vitro enzyme reaction,
CHS also produces bis-noryangonin (BNY) and 4-couma-
the shunt products of the minimal type II PKS from
(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,
S.; K a¨ rcher, B.; Schr o¨ der, G.; Schr o¨ der, J. J. Biol. Chem. 1995, 270, 7922-
7928. (f) Suh, D. Y.; Fukuma, K.; Kagami, J.; Yamazaki, Y.; Shibuya, M.;
Ebizuka, Y.; Sankawa, U. Biochem. J. 2000, 350, 229-235. (g) Jez, J. M.;
Bowman, M. E.; Noel, J. P. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5319-
5324. (h) Austin, M. B.; Bowman, M. E.; Ferrer, J.-L.; Schr o¨ der, J.; Noel,
J. P. Chem. Biol. 2004, 11, 1179-1194. (i) Abe, I.; Sano, Y.; Takahashi,
Y.; Noguchi, H. J. Biol. Chem. 2003, 278, 25218-25226.
2
†
University of Shizuoka.
PRESTO, Japan Science and Technology Agency.
Mitsubishi Kagaku Institute of Life Sciences.
§
‡
(
1) For recent reviews, see: (a) Schr o¨ 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.
1
0.1021/ol052912h CCC: $33.50
© 2006 American Chemical Society
Published on Web 01/07/2006

Figure 1. Formation of (A) chalcone by CHS, (B) SEK4/SEK4b by OKS (and CHS S338V), and (C) other polyketide products.
4
Streptomyces coelicolor and the longest polyketides gener-
previous papers, we reported that the residue 197 determines
the polyketide chain length and product specificities in the
octaketide-producing A. arborescens OKS and the penta-
3
ated by the structurally simple type III PKS. In A. arbore-
scens OKS, the CHS’s active-site residues Thr197, Gly256,
and Ser338 (numbering in Medicago satiVa CHS ) are
2
a
3
ketide-producing A. arborescens PCS.
uniquely replaced with Gly, Leu, and Val, respectively
(
To further study the structure-function relationship be-
T197G/G256L/S338V).3b Interestingly, the three residues
tween CHS and OKS enzyme, here we constructed a series
7
lining the active-site cavity are sterically altered in a number
of functionally divergent type III PKSs including A. arbore-
scens pentaketide chromone synthase (PCS) (T197M/G256L/
of Scutellaria baicalensis CHS mutants in which the three
residues were changed from those in CHS to those in OKS
(T197G, G256L, and S338V), and investigated the mecha-
nistic consequences of the mutations using 4-coumaloyl-CoA
and/or malonyl-CoA as substrates.
Interestingly, in the absence of the coumaroyl starter, both
wild-type and the mutant S. baicalensis CHSs initiated
decarboxylative condensation of malonyl-CoA, but most of
the polyketide chain elongation reactions were terminated
at the triketide stage to predominantly produce triacetic acid
lactone (TAL) (Figure 2A). This is in good agreement with
an earlier report that mutation of M. satiVa CHS at the
residues (T197L, G256L, and S338I) resulted in functional
3a
S338V) , Rheum palmatum aloesone synthase (T197A/
5
G256L/S338T), and Gerbera hybrida 2-pyrone synthase
6
(
2PS) (T197L/G256L/S338I). These chemically inert resi-
dues have been shown to control starter substrate and product
specificity by steric modulation of the active-site cavity in
2d,6b
M. satiVa CHS and in G. hybrida 2PS.
Further, in
(3) (a) Abe, I.; Utsumi, Y,; Oguro, S.; Morita, H.; Sano, Y.; Noguchi,
H. J. Am. Chem. Soc. 2005, 127, 1362-1363. (b) Abe, I.; Oguro, S.; Utsumi,
Y,; Sano, Y.; Noguchi, H. J. Am. Chem. Soc. 2005, 127, 12709-12716.
(4) (a) Fu, H.; Ebert-Khosla, S.; Hopwood, D. A.; Khosla, C. J. Am.
Chem. Soc. 1994, 116, 4166-4170. (b) Fu, H.; Hopwood, D. A.; Khosla,
C. Chem. Biol. 1994, 1, 205-210.
(
5) (a) Abe, I.; Utsumi, Y,; Oguro, S.; Noguchi, H. FEBS Lett. 2004,
(7) (a) Abe, I.; Morita, H.; Nomura, A.; Noguchi, H. J. Am. Chem. Soc.
2000, 122, 11242-11243. (b) The deduced amino acid sequences of S.
baicalensis CHS showed 77.4% (302/389) identity with those of M. satiVa
CHS and 60.4% (235/389) identity with A. arboresens OKS. The
recombinant enzyme with an additional hexahistidine tag at the C-terminal
was expressed in E. coli and purified by Ni-chelate chromatography as
5
2
62, 171-176. (b) Abe, I.; Watanabe, T.; Lou, W.; Noguchi, H. FEBS J.
006, 273, 208-218.
(6) (a) Eckermann, S.; Schr o¨ der, G.; Schmidt, J.; Strack, D.; Edrada, R.
A.; Helariutta, Y.; Elomaa, P.; Kotilainen, M.; Kilpel a¨ inen, I.; Proksch, P.;
Teeri, T. H.; Schr o¨ der, J. Nature 1998, 396, 387-390. (b) Jez, J. M.; Austin,
M. B.; Ferrer, J.; Bowman, M. E.; Schr o¨ der, J.; Noel, J. P. Chem. Biol.
7a
described before. The wild-type enzyme showed KM ) 36.1 µM and kcat
-
1
2
000, 7, 919-930.
) 1.26 min for 4-coumaroyl-CoA.
500
Org. Lett., Vol. 8, No. 3, 2006

Figure 3. Schematic representation of the active-site architecture
of (A) CHS, (B) CHS S338V mutant, and (C) A. arborescens OKS.
(D) CHS’s active-site cavity and an entrance to a novel buried
pocket, and (E) the entrance of the buried pocket (a putative
polyketide elongation tunnel) that extends into the “floor” of the
active-site cavity.
Figure 2. Distribution pattern of polyketides produced by CHS
mutants: (A) from malonyl-CoA (all products), (B) from malonyl-
CoA (pentaketides and octaketides), (C) from 4-coumaroyl-CoA/
malonyl-CoA. (1) wild-type, (2) T197G, (3) G256L, (4) S338V,
(5) G256L/S338V, (6) T197G/G256L, (7) T197G/S338V, (8)
T197G/G256L/S338V, (9) T197A/G256L/S338V, (10) T197M/
G256L/S338V.
G256L and G256L/S338V mutant did not afford any
products from 4-coumaroyl-CoA (Figure 2C).
6b
conversion into a TAL-producing enzyme. However, very
surprisingly, careful examination of the enzyme reaction
products revealed that CHS S338V mutant yielded a trace
amount of SEK4/SEK4b in addition to 5,7-dihydroxy-2-
Out of the three point mutations tested (T197G, G256L,
and S338V), the hydrophobic replacement S338V is critical
for production of the longer chain polyketides including
8
SEK4/SEK4b. In contrast, T197G and G256L mutant did
3a
3b
methylchromone, 2,7-dihydroxy-5-methylchromone, and
tetracetic acid lactone3b (Figures 1C and 2B). It was thus
for the first time demonstrated that CHS could be engineered
to produce longer octaketides by the single amino acid
replacement. Furthermore, the SEK4/SEK4b-forming activity
was dramatically increased in an OKS-like triple mutant
not produce any malonate-derived polyketides except TAL
and tetraacetic acid lactone. On the basis of the published
X-ray crystal structure of M. satiVa CHS at 1.56 Å
2
a
resolution, the residue 338 is located in proximity of the
catalytic Cys164 at the “ceiling” of the active-site cavity and
plays a crucial role in the polyketide chain elongation
(
T197G/G256L/S338V) (Figure 2B). On the other hand, in
the presence of 4-coumaroyl-CoA in the assay mixture, most
of the S. baicalensis CHS mutants still accepted the cou-
maroyl starter to produce chalcone, whereas interestingly
(8) S. baicalensis CHS S338I (2PS-like) mutant also produced a trace
amount of SEK4/SEK4b, whereas S338A, S338F, and S338T (ALS-like)
mutant only produced the pentaketide chromones. Interestingly, S338C
mutant did not produce any malonate-derived polyketides except TAL.
Org. Lett., Vol. 8, No. 3, 2006
501

reactions. Further interestingly, the crystal structure also
revealed presence of an additional buried pocket that extends
pocket,2a thus controlling the starter substrate selectivity in
S. baicalensis CHS (Figure 3). In fact, G256L and G256L/
S338V mutant did not yield any polyketide products from
the bulky coumaroyl starter (Figure 2C), but instead ef-
ficiently produced TAL from the malonyl starter (Figure 2A).
It was interesting that the additional, the downward expand-
ing, T197G replacement recovered chalcone-forming activity
of the CHS mutants (T197G/G256L and T197G/G256L/
S338V) (Figure 2C).
into the “floor” of the traditional CHS active site (Figure
9
3
). Presumably, the S338V mutaion provided steric guidance
so that the linear polyketide intermediate extends into the
buried pocket, thereby leading to formation of the longer
polyketides (Figure 3). Most of the polyketide elongation
reactions were, however, terminated at the triketide stage to
yield TAL as a predominant product. A similar active-site
architecture with a downward expanding polyketide tunnel
has been recently reported for a bacterial pentaketide-
producing type III PKS, 1,3,6,8-tetrahydroxynaphthalene
synthase, from S. coelicolor that shares only ca. 20% amino
acid sequence identity with CHS.9
In summary, it was for the first time demonstrated that
the CHS active-site can be extended to allow for the synthesis
of larger polyketides including SEK4/SEK4b by a single
amino acid substitution S338V. Further, the octaketide-
forming activity was dramatically increased in the OKS-like
S. baicalensis 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. These results provided structural basis for
understanding the functional diversity of type III PKS
enzymes and suggest strategies for engineered biosynthesis
of plant polyketides.
On the other hand, Thr197 in S. baicalensis CHS functions
as a gate keeper at the entrance of the buried pocket along
with Gly211, Gln212, Leu263, Thr264, and Phe265 (Figure
3E). The replacement of Thr197 with less bulky Gly in
T197G/S338V and T197G/G256L/S338V mutant widely
opens the gate, thereby expanding the putative polyketide
chain elongation tunnel, which led to significantly increased
production of the longer chain polyketides including the
octaketide SEK4/SEK4b (Figure 2A and 2B). Whereas,
small-to-large substitutions in place of the residue 197
Acknowledgment. This work was in part supported by
the COE21 Program, Grant-in-Aid for Scientific Research
(T197A/G256L/S338V and T197M/G256L/S338V) resulted
(nos. 16510164, 17310130, and 17035069), Cooperation of
in decrease of the octaketide-forming activity and the
concomitant formation of shorter chain polyketides (Figure
Innovative Technology and Advanced Research in Evolu-
tional Area (CITY AREA), from the MEXT, Japan, and by
Health and Labor Sciences Research Grant from the MHLW,
Japan.
2A and 2B), which is well consistent with our previous
reports that the residue 197 determines the polyketide chain
3b
length and product specificities in A. arborescens OKS and
3
a
A. arborescens PCS.
Finally, the OKS- and 2PS-like bulky G256L substitution6b,9
Supporting Information Available: Experimental pro-
cedures. This material is available free of charge via the
Internet at http://pubs.acs.org.
contributes to a steric constriction of the coumaroyl binding
(
9) Austin, M. B.; Izumikawa, M.; Bowman, M. E.; Udwary, D. W.;
Ferrer, J.-L.; Moore, B. S.; Noel, J. P. J. Biol. Chem. 2004, 279, 45162-
5174.
4
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Org. Lett., Vol. 8, No. 3, 2006