Enzymatic formation of an aromatic dodecaketide by engineered plant
Kiyofumi Wanibuchia,b, Hiroyuki Moritaa, Hiroshi Noguchib, Ikuro Abea,⇑
aGraduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
bSchool of Pharmaceutical Sciences, University of Shizuoka and Global COE Program, Shizuoka 422-8526, Japan
a r t i c l ei n f o
Received 14 January 2011
Revised 27 January 2011
Accepted 31 January 2011
Available online 3 February 2011
Pentaketide chromone synthase
a b s t r a c t
Octaketide synthase (OKS) from Aloe arborescens is a plant-specific type III polyketide synthase (PKS) that
catalyzes iterative condensations of eight molecules of malonyl-CoA to produce the C16aromatic octake-
tides SEK4 and SEK4b. On the basis of the crystal structures of OKS, the F66L/N222G double mutant was
constructed and shown to produce an unnatural dodecaketide TW95a by sequential condensations of 12
molecules of malonyl-CoA. The C24naphthophenone TW95a is a product of the minimal type II PKS (whiE
from Streptomyces coelicolor), and is structurally related to the C20decaketide benzophenone SEK15, the
product of the OKS N222G point mutant. The C24dodecaketide naphthophenone TW95a is the first and
the longest polyketide scaffold generated by a structurally simple type III PKS. A homology model pre-
dicted that the active-site cavity volume of the F66L/N222G mutant is increased to 748 Å3, from
652 Å3of the wild-type OKS. The structure-based engineering thus greatly expanded the catalytic reper-
toire of the simple type III PKS to further produce larger and more complex polyketide molecules.
? 2011 Elsevier Ltd. All rights reserved.
The chalcone synthase (CHS) (EC 220.127.116.11) superfamily of type
III polyketide synthases (PKSs) are structurally and mechanistically
simple enzymes.1–3In contrast to the type I (modular type) and
type II (subunit type) PKSs of the megaenzyme systems, the struc-
turally simple type III PKSs accept free CoA thioesters as substrates
without the involvement of an acyl carrier protein, and perform
iterative condensation and cyclization reactions to produce an ar-
ray of chemically and structurally divergent polyphenol scaffolds.
As we previously reported, octaketide synthase (OKS) from Aloe
arborescens is a plant-specific type III PKS that catalyzes iterative
condensations of eight molecules of malonyl-CoA to yield a 1:4
mixture of the aromatic octaketides SEK4 and SEK4b (Scheme 1A
and Fig. 1A).4The C16aromatic octaketides are the products of
the minimal type II PKS for the benzoisochromanequinone actino-
rhodin (act from Streptomyces coelicolor).5,6Furthermore, we re-
ported that the substitution of the active-site Asn222 of OKS
with Gly yielded a mutant that catalyzes the formation of the C20
decaketide benzophenone SEK15, by the condensation of ten mol-
ecules of malonyl-CoA (Scheme 1B and Fig. 1B).7The decaketide
benzophenone was previously reported as a product of genetically
engineered type II PKSs.5The octaketide-forming OKS thus gained
the decaketide synthase activity by the simple steric modulation of
a single, chemically inert, residue lining the active-site cavity. In-
deed, the crystal structures of the wild-type and N222G mutant en-
zymes revealed that the large-to-small substitution increased the
volume of the active-site cavity to 693 Å3, from 652 Å3of the
wild-type OKS (Fig. 2A and B).1,8
To further manipulate the OKS enzyme reaction, we expanded
the polyketide elongation tunnel of the N222G mutant by simulta-
neously substituting the neighboring Phe66 residue, at the bottom
of the tunnel, with less bulky amino acids. Thus, we constructed a
set of A. arborescens OKS double mutants (F66G/N222G, F66A/
investigated the mechanistic consequences of the mutations.9A
homology model based on the N222G mutant predicted that the
active-site cavity volume is increased to 748 Å3in the F66L/
N222G double mutant (Fig. 2C).
The double mutants were heterologously expressed in Esche-
richia coli as His-tagged fusion proteins, at similar levels to the
wild-type enzyme, and were purified to homogeneity, as previ-
ously reported for the wild-type and N222G mutant enzymes.10
When incubated with malonyl-CoA as a substrate, the F66V/
N222G and F66S/N222G mutants displayed enzyme activities sim-
ilar to that of the N222G point mutant, and efficiently produced the
decaketide benzophenone SEK15, whereas the F66G/N222G and
F66A/N222G mutants completely lost the enzyme activity.11Nota-
bly, the OKS F66G point mutant also lacked the enzyme activity,
and the F66A mutant showed a notable decrease of the activity.
These results suggested that the substitution of Phe66 with the
0960-894X/$ - see front matter ? 2011 Elsevier Ltd. All rights reserved.
Abbreviations: OKS, Octaketide synthase; PCS, Pentaketide chromone synthase;
CHS, Chalcone synthase; PKS, Polyketide synthase.
⇑Corresponding author. Tel.: +81 3 5841 4740; fax: +81 3 5841 4744.
E-mail address: firstname.lastname@example.org (I. Abe).
Bioorganic & Medicinal Chemistry Letters 21 (2011) 2083–2086
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small Gly or Ala residue causes significant conformational changes
in the folding of the enzyme, which lead to the loss of the enzyme
On the other hand, the major product generated by the F66L/
N222G double mutant was obtained in 6% yield (calculated by an
incubation with [2-14C]malonyl-CoA12as the substrate under the
standard assay conditions), in addition to the decaketide SEK15
as a minor product (Fig. 1C). The major product gave a UV spec-
trum (kmax 234, 284, 340, and 402 nm) and a parent ion peak
[M+H]+at m/z 451 on LC-ESIMS, indicating the formation of a prod-
uct of 12 condensations of malonyl-CoA. The spectroscopic data
(LC-ESIMS, UV, and1H NMR) of the product obtained from a large
scale incubation were quite similar to those of the C20benzophe-
none SEK15, with the 4-hydroxy-2-pyrone and 2,4-hydroxy-6-
methylphenyl ketone functionalities.13However, the
spectrum revealed the presence of five aromatic protons (d 7.01,
6.59, 6.40, 6.18, and 6.14), indicating the presence of a naphth-
ophenone, instead of the benzophenone moiety. Thus, the spectro-
scopic data were identical to those of the previously reported C24
dodecaketide naphthophenone TW95a, which is a product of the
minimal type II PKS (whiE from S. coelicolor),14and is structurally
related to the C20decaketide benzophenone SEK15, the product
of the OKS N222G point mutant.7This is the first demonstration
of C24dodecaketide production by a structurally simple type III
A steady-state kinetics analysis revealed a KM= 32 lM and a
kcat= 4.7 ? 10?2min?1for malonyl-CoA by the OKS F66L/N222G
double mutant with respect to the TW95a-forming activity, with
a pH optimum at 6.5.15On the other hand, the previously reported
SEK15-forming OKS N222G mutant exhibited a KM= 55 lM and a
kcat= 2.7 ? 10?3min?1, and the SEK4b-forming wild-type OKS
showed a KM= 94 lM and a kcat= 9.4 ? 10?2min?1. Notably, the
enzyme activities of the N222G and F66L/N222G mutants are com-
parable to that of the wild-type OKS, and the KMvalues suggest
even higher affinity for the substrate binding to the active-site, de-
spite the lower kcatvalues.
It is remarkable that the OKS F66L/N222G mutant not only cat-
alyzed the condensation of 12 molecules of malonyl-CoA but also
attained the naphthalene ring forming activity to produce the C24
dodecaketide naphthophenone TW95a, which is the first and the
longest polyketide scaffold generated by a structurally simple type
III PKS. As mentioned above, a homology model predicted that the
active-site cavity volume of the dodecaketide-producing N222G/
F66L double mutant is increased to 748 Å3from 693 Å3of the
decaketide-producing N222G point mutant, and from 652 Å3of
the octaketide-producing wild-type OKS. Thus, the replacement
of the two residues, Phe66 and Asn222, expanded the active-site
cavity, thus extending the number of condensation reactions and
the resulting polyketide chain length. It is remarkable that the
functional conversion appeared to be caused by the simple steric
modulation of the active-site, accompanied by the conservation
of the Cys-His-Asn catalytic triad. This was also the case for the
previously reported A. arborescens pentaketide chromone synthase
(PCS), which produces 5,7-dihydroxy-2-methylchromone from five
molecules of malonyl-CoA.16,17The structure-based PCS F80A/
Y82A/M207G triple mutant, with an expanded active-site cavity,
Scheme 1. Proposed mechanism for the formation of (A) SEK4 and SEK4b by the wild-type OKS, (B) SEK 15 by the N222G mutant OKS, and (C) TW95a by the F66L/N222G
K. Wanibuchi et al./Bioorg. Med. Chem. Lett. 21 (2011) 2083–2086
01020 30 min
01020 30 min
Figure 1. HPLC elution profiles of the enzyme reaction products of (A) the wild-type OKS, (B) the N222G mutant OKS, and (C) the F66L/N222G mutant OKS.
H316 H316 H316
Y82 Y82 Y82
Figure 2. Surface and schematic representations of the active-site architectures of the wild-type and mutant OKSs. Crystal structure of (A) the wild-type OKS, and (B) the
N222G mutant OKS, and homology model of (C) the F66L/N222G mutant OKS. The substrate entrances are indicated with arrows. The bottoms of the active-site are indicated
by purple surfaces. The Cys-His-Asn catalytic triads are shown as yellow stick models. The mutated residues Asn222 (Gly222) and Phe66 (Leu66) are highlighted as blue and
purple stick models, respectively.
K. Wanibuchi et al./Bioorg. Med. Chem. Lett. 21 (2011) 2083–2086
was shown to produce the unnatural novel nonaketide naphthopy- Download full-text
rone by sequential condensations of nine molecules of malonyl-
The proposed mechanism for the formation of the dodecaketide
naphthophenone TW95a by the F66L/N222G mutant closely paral-
lels the case of the decaketide SEK15 produced by the N222G mu-
tant, andinvolves consecutive
condensations and terminal a-pyrone ring formation (Scheme 1B
and C). Presumably, the enzyme catalyzes the first aromatic ring
formation reaction at the middle of the polyketide intermediate
during the sequential decarboxylative condensations of 12 mole-
cules of malonyl-CoA. Thus, dual C–C bond formation at C-16/C-7
and C-14/C-9 generates the naphthalene ring system of the dode-
caketide TW95a, whereas a single aldol-type cyclization produces
the benzene ring of the decaketide SEK15. Alternatively, the aro-
matic ring formation is possibly initiated at the methyl end of
the elongating polyketide intermediate, as in the case for the for-
mation of the octaketide SEK4b by the wild-type OKS. The partially
cyclized aromatic intermediates would then be released from the
active-site by the formation of the terminal a-pyrone ring, which
could be an important process for the release of the polyketide
products from the thioester-linked active-site Cys residue. Finally,
it should be noted that the formation of the dodecaketide naphth-
ophenone by the A. arborescens OKS mutant suggests further
involvement of the CHS-superfamily type III PKSs in the biogenesis
of the anthrone and anthraquinone scaffolds in the aloe plant.4
In summary, this is the first demonstration of the formation of a
dodecaketide by a structurally simple type III PKS. The structure-
based engineering thus greatly expanded the catalytic repertoire
of the simple CHS superfamily type III PKS, to produce larger, more
complex polyketide molecules.
We thank Dr. Yukihiro Goda (National Institute of Health Sci-
ences) for NMR measurement of TW95a. This work was supported
in part by a Grant-in-Aid for Scientific Research from the Ministry
of Education, Culture, Sports, Science and Technology, Japan (to I.A.
and H.M.), and grants from The Naito Foundation (to I.A.), The Cos-
metology Research Foundation (to I.A.), and the Uehara Memorial
Foundation (to H.M.). K.W. is a recipient of the JSPS Fellowship
for Young Scientists.
References and notes
1. Abe, I.; Morita, H. Nat. Prod. Rep. 2010, 27, 809.
2. Austin, M. B.; Noel, J. P. Nat. Prod. Rep. 2003, 20, 79.
3. Comprehensive Natural Products Chemistry; Schröder, J., Ed.; Elsevier: Oxford,
1999; 1, p 749.
4. Abe, I.; Oguro, S.; Utsumi, Y.; Sano, Y.; Noguchi, H. J. Am. Chem. Soc. 2005, 127,
5. Fu, H.; Ebert-Khosla, S.; Hopwood, D. A.; Khosla, C. J. Am. Chem. Soc. 1994, 116,
6. Fu, H.; Hopwood, D. A.; Khosla, C. Chem. Biol. 1994, 1, 205.
7. Shi, S.-P.; Wanibuchi, K.; Morita, H.; Endo, K.; Noguchi, H.; Abe, I. Org. Lett.
2009, 11, 551.
8. Morita, H.; Kondo, S.; Kato, R.; Wanibuchi, K.; Noguchi, H.; Sugio, S.; Abe, I.;
Kohno, T. Acta Crystallogr., Sect. F 2007, 63, 947.
9. Site-directed mutagenesis:The plasmids expressing Aloe arborescens OKS double
mutants (F66G/N222G, F66A/N222G, F66V/N222G, F66S/N222G, and F66L/
N222G) were constructed with a QuikChange Site-Directed Mutagenesis Kit
(Stratagene), according to the manufacturer0s protocol, using the following
pairs of primers (mutated codons are underlined) and the previously reported
plasmid bearing the N222G OKS mutant as the template: F66G (50-
TCGCAT-30and 50-TGTCTTTTTGCAGATGCGATCGGCCTTCTTCTTGAG-30), F66V
(50-GGTCGAGCTCAAGAAGAAGGTCGATCGCAT-30) and (50-TGTCTTTTTGCAGAT
GCGATCGACCTTCTTCTTGAG-30), F66S (50-GGTCGAGCTCAAGAAGAAGTCGGATC
10. Expression and purification: After confirmation of the sequence, the plasmid was
transformed into E. coli BL21(DE3)pLysS. The cells harboring the plasmid were
cultured to an A600of 0.6 in LB medium containing 100 lg/mL ampicillin at
37 ?C. Subsequently, 1.0 mM isopropylthio-b-D-alactopyranoside was added to
induce protein expression, and the cells were further cultured at 23 ?C for 16 h.
All of the following procedures were performed at 4 ?C. The E. coli
BL21(DE3)pLysS cells transformed with the OKS mutants were harvested by
centrifugation and resuspended in 40 mM potassium phosphate buffer, pH 7.9,
containing 0.1 M NaCl and 5 mM imidazole. The cells were disrupted by
sonication and centrifuged at 10,000g for 30 min. The supernatant was passed
through a Ni Sepharose™ 6 Fast Flow column (GE Healthcare). After the
column was washed with 20 mM potassium phosphate buffer, pH 7.9,
containing 0.5 M NaCl and 40 mM imidazole, the recombinant enzyme was
finally eluted with 15 mM potassium phosphate buffer, pH 7.5, containing 10%
glycerol and 500 mM imidazole. The recombinant PKSs were fairly soluble, and
the Ni-chelate affinity column chromatography afforded 3–5 mg of pure
enzymes from 1 L of the E. coli culture. The protein concentration was
determined by the Bradford method (Protein Assay Dc, Bio-Rad, Hercules, CA,
USA) using bovine gamma globulin as the standard.
11. Enzyme reaction: The standard reaction mixture contained 216 nmol of
malonyl-CoA and 10 lg of the purified recombinant enzyme in a final
volume of 500 lL of 100 mM potassium phosphate buffer, pH 6.5. Reactions
were incubated at 30 ?C for 16 h. The products were then extracted with 2 mL
of ethyl acetate. The products were separated by reverse-phase HPLC (JASCO
880) on a TSK-gel ODS-80Ts column (4.6 ? 150 mm, TOSOH), at a flow rate of
0.8 mL/min. Gradient elution was performed with H2O and MeOH, both
containing 0.1% TFA: 0–5 min, 30% MeOH; 5–17 min, linear gradient from 30%
to 60% MeOH; 17–25 min, 60% MeOH; 25–27 min, linear gradient from 60% to
70% MeOH. Elutions were monitored by a multichannel UV detector (MULTI
340, JASCO) at 280 nm; UV spectra (200–400 nm) were recorded every 0.4 s.
Online LC-ESIMS spectra were measured with an Agilent Technologies HPLC
1100 series HPLC coupled to a Bruker Daltonics Esquire4000 ion trap mass
spectrometer fitted with an ESI source. HPLC separations were performed
under the same conditions as described above. The ESI capillary temperature
and the capillary voltage were 320 ?C and 4.0 V, respectively. The tube lens
offset was set at 20.0 V. All spectra were obtained in the positive mode, over a
mass range of 50–600 m/z, and at a range of one scan every 0.2 s. The collision
gas was helium, and the relative collision energy scale was set at 30.0%
(1.5 eV). The samples for the LC-HRMS were analyzed with an Agilent 1100
12. [2-14C]Malonyl-CoA was purchased from GE Healthcare.
1H), 6.14 (s, 1H), 5.70 (s, 1H), 5.18 (d, 1H), 3.67 (s, 2H), 1.92 (s, 3H); UV kmax
234, 284, 340 and 402 nm; ESI-MS: m/z 451 [M+H]+; ESI-MS/MS: 327, 261, 191,
151; HRMS (TOF) found for [C24H19O9]+451.1006, calcd 451.1007. The data
were identical to those previously reported in the literature.14
14. Yu, T.-W.; Shen, Y.; McDaniel, R.; Floss, H. G.; Khosla, C.; Hopwood, D. A.; Moor,
B. S. J. Am. Chem. Soc. 1998, 120, 7749.
15. Enzyme kinetics. Steady-state kinetic parameters were determined by using
[2-14C]malonyl-CoA (1.8 mCi/mmol) as the substrate. The experiments were
performed in triplicate using five concentrations of malonyl-CoA (108, 43.2,
21.6, 10.8 and 4.3 lM) in the assay mixture, containing 108 lM of malonyl-
CoA, 3 lg of purified enzyme, and 1 mM EDTA, in a final volume of 500 lL of
100 mM potassium phosphate buffer, pH 6.5. The reactions were incubated at
30 ?C for 120 min. The reaction products were extracted and separated by TLC
(Merck Art. 1.11798 Silica gel 60 F254; ethyl acetate/hexane/AcOH = 63:27:5,
v/v/v). Radioactivities were quantified by autoradiography using a bioimaging
analyzer BAS-2000II (FUJIFILM). Lineweaver–Burk plots of data were employed
to derive the apparent KMand kcatvalues (average of triplicates), using the
ENZFITTER software (BIOSOFT).
16. Abe, I.; Utsumi, Y.; Oguro, S.; Morita, H.; Sano, Y.; Noguchi, H. J. Am. Chem. Soc.
2005, 127, 1362.
17. Morita, H.; Kondo, S.; Oguro, S.; Noguchi, H.; Sugio, S.; Abe, I.; Kohno, T. Chem.
Biol. 2007, 14, 359.
18. Abe, I.; Morita, H.; Oguro, S.; Noma, H.; Wanibuchi, K.; Kawahara, N.; Goda, Y.;
Noguchi, H.; Kohno, T. J. Am. Chem. Soc. 2007, 129, 5976.
1H NMR (800 MHz, DMSO-d6) d 7.01 (s, 1H), 6.59 (d, 1H), 6.40 (d, 1H), 6.18 (s,
K. Wanibuchi et al./Bioorg. Med. Chem. Lett. 21 (2011) 2083–2086