2152
S.-P. Shi et al. / Tetrahedron Letters 50 (2009) 2150–2153
the thioester-linked active-site cysteine.5e The results suggested
OKS
OH
6 xCoAS
CoAS
that manipulation of the enzyme reactions by combination of the
precursor-directed biosynthesis and structure-guided engineering
of the enzyme would lead to further production of unnatural novel
polyketide scaffolds.
N222G
+
O
O
O
benzoyl-CoA
O
malonyl-CoA
OH
OH
O
OH
O
O
O
HO
OH
HO
Acknowledgments
CH3
O
O
O
O
O
O
HO
O
HO
This work was supported by the PRESTO program from Japan
Science and Technology Agency, Grant-in-Aid scientific Research
from the Ministry of Education, Culture, Sports, Science and Tech-
nology, Japan, and Research Grants from the Astellas Foundation
for Research on Metabolic Disorders, and the Uehara Memorial
Foundation, Japan. S.-P.S. and K.W. are recipients of the JSPS Post-
doctoral Fellowship for Foreign Researchers (no. P07048) and the
JSPS Fellowship for Young Scientists (no. 207112), respectively.
O
SEnz
6
SEK 15
C20
C19
O
O
OH
3 xCoAS
+
O
O
O
OH
7
Scheme 3. Enzymatic formation of
a
C19 heptaketide benzophenone and
a
tetraketide pyrone by OKS N222G mutant (from the benzoyl starter).
Supplementary data
Supplementary data (experimental details and a complete set of
NMR data and charts are provided) associated with this article can
protons and carbons were completely assigned by NMR (1H and 13
NMR, HMQC, and HMBC) spectroscopy.13–15
C
For the product 3, its ESIMS spectrum indicated loss of a typical
ion peak 126, which is a characteristic fragment for the 4-hydroxy-
6-methylpyran-2-oxo moiety. Unfortunately, the structural eluci-
dation of 3 by NMR failed because this compound is very unstable,
and spontaneously and completely changed into a C19 heptaketide
(3a) (0.5 mg, 5% yield), whose structure was unambiguously eluci-
dated by NMR (1H and 13C NMR, HMQC, and HMBC) and MS spec-
troscopy.16 Considering the ESIMS data and biogenetic reasoning, it
is likely that 3a was produced from the putative intermediate 3 via
opening of the terminal pyrone ring, decarboxylation, and recycli-
zation (Scheme 2B). Similar decarboxylative conversion of a keto 2-
pyrone to a 4-pyrone has been also reported in a literature.12
In contrast, the smaller C6–C1 benzoyl-CoA did not fit well into
the active-site of wild-type A. arborescens OKS. When the enzyme
was incubated with benzoyl-CoA and malonyl-CoA as substrates,
most of the enzyme reactions were initiated by malonyl-CoA,
and the octaketides SEK4 and SEK4b were obtained as dominant
products (Fig. 1C). On the other hand, however, the structure-
guided N222G mutant with the expanded active-site cavity ac-
cepted the benzoyl starter and carried out six condensations with
malonyl-CoA to produce a novel C19 heptaketide benzophenone (6)
(0.8 mg, 8% yield) as well as a tetraketide 4-hydroxy-6-(2-oxo-2-
phenylethyl)-2-pyrone (7)3b (0.6 mg, 7% yield) (Fig. 1D and Scheme
3). The structures were unambiguously established by NMR (1H
and 13C NMR, HMQC, and HMBC) and MS spectroscopy.17 Here it
should be noted that the structure of the C19 heptaketide 6 showed
close similarity to that of the C20 decaketide SEK15 produced from
10 molecules of malonyl-CoA by the N222G mutant (Scheme 1C).
In summary, the present work describes the enzymatic forma-
tion of six novel polyketides from phenylacetyl-CoA and benzoyl-
CoA by a plant-specific type III PKS. It is remarkable that wild-type
OKS and the structure-guided N222G mutant afforded significantly
different product pattern; the wild-type enzyme produced C16 pen-
taketide coumarin, whereas the point mutant dramatically ex-
tended the product chain length up to the C22 octaketides which
are currently the longest polyketides generated by the structurally
simple type III PKSs. Further, the C6–C2 phenylacetyl starter better
fits into the active-site of the enzymes and more efficiently initi-
ated the sequential condensation reactions than the C6–C1 benzoyl
starter. In all the cases, the enzymes catalyzed the chain elongation
and the first aromatic ring formation at the middle of the polyke-
tide intermediates as in the case of the production of SEK4/SEK4b
References and notes
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7. Steady state kinetic analysis revealed that KM = 91.6
for phenylacetyl-CoA for the C16 coumarin forming activity.
l
M and kcat = 0.022 minÀ1
}
}
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(400 MHz, CD3OH): d 7.10–7.26 (m, 5H), 6.61 (br s, 1H), 6.56 (br s, 1H), 5.38 (s,
1H), 4.46 (s, 2H). 13C NMR (100 MHz, CD3OH): d 171.7, 166.4, 162.5, 158.9,
143.2, 142.2, 129.6 (2Â), 129.2 (2Â), 126.8, 117.8, 107.9, 102.2, 89.0, 41.4.
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and SEK15. Formation of the terminal
a-pyrone ring could be an
important process for the release of the polyketide products from