Potent oligomerization and macrocyclization activity of the thioesterase domain of vicenistatin polyketide synthase

Article abstract of DOI:10.1055/s-0031-1290385

The thioesterase domain of the polyketide synthase involved in the biosynthesis of the 20-membered macrolactam antibiotic vicenistatin (VinTE) was found to catalyze oligomerization and macrocyclization of ω-hydroxy fatty acid ethyl esters to afford 17-28-membered macrocyclic lactones. The ring sizes of the macrocycles appear to be limited to the more moderate sizes because of the space limitation of the active site of VinTE. It was also verified that the initially formed linear dimer is first released from the active site of VinTE and then is recognized again by VinTE prior to its transformation to the cyclic dimer.

Full text of DOI:10.1055/s-0031-1290385

LETTER
▌1843
letter
Potent Oligomerization and Macrocyclization Activity of the Thioesterase
Domain of Vicenistatin Polyketide Synthase
Oligomerization and Macrocyclization by VinTE
Fumitaka Kudo,a Yusaku Asou,a Moe Watanabe,a Takashi Kitayama,b Tadashi Eguchi*b
a
Department of Chemistry, Tokyo Institute of Technology, 2-12-1 O-okayama, Meguro-ku, Tokyo 152-8551, Japan
b
Department of Chemistry and Materials Science, Tokyo Institute of Technology, 2-12-1 O-okayama, Meguro-ku, Tokyo 152-8551, Japan
Fax +81(3)57342631; E-mail: eguchi@cms.titech.ac.jp
Received: 01.02.2012; Accepted after revision: 27.04.2012
cysteamine (NAC) thioester and phenyl thioester (a mim-
Abstract: The thioesterase domain of the polyketide synthase in-
ic of ACP-phosphopantetheine thioester),13 VinTE also
volved in the biosynthesis of the 20-membered macrolactam antibi-
recognizes the simple ethyl ester of the seco-amino acid of
otic vicenistatin (VinTE) was found to catalyze oligomerization and
macrocyclization of ω-hydroxy fatty acid ethyl esters to afford 17–
the aglycon vicenilactam in addition to the NAC thioester
28-membered macrocyclic lactones. The ring sizes of the macrocy- to form a macrolactam.6 Only VinTE and TyrC TE4 are
cles appear to be limited to the more moderate sizes because of the
space limitation of the active site of VinTE. It was also verified that
known to recognize ester substrates. Thus we prepared a
more stable ethyl ester substrate rather than NAC thioester
the initially formed linear dimer is first released from the active site
for the examination of specificity.
of VinTE and then is recognized again by VinTE prior to its trans-
formation to the cyclic dimer.
ACP
S
H
HO
N
Key words: macrocycles, cyclization, oligomerization, polyketide
synthase, thioesterase domain
HO
NH2
VinTE
O
O
vicenilactam
Macrocyclic natural products such as macrolide
polyketide antibiotics (e.g., erythromycin, avermectin)
and nonribosomal peptide antibiotics (e.g., gramicidin,
daptomycin) are biosynthesized by polyketide synthases
(PKS) and nonribosomal peptide synthetases (NRPS), re-
spectively. In general, the C-terminal thioesterase (TE)
domain of PKS and NRPS is responsible for the macrocy-
clizations.1,2 The TE domain catalyzes the cleavage of the
thioester bond of the elongated polyketide-acyl carrier
protein (ACP) or polypeptide-peptidyl carrier protein
(PCP) via a catalytic active-site serine residue, and the
subsequent intramolecular nucleophilic attack of a hy-
droxy or amino group to form a macrocycle. Walsh et al.
have demonstrated the synthetic potential of the truncated
NRPS-TE domains by showing that they can catalyze the
formation of various unnatural macrocyclic polypep-
tides.3–5 In addition, several truncated PKS TE domains
including vicenistatin PKS,6 picromicin PKS,7,8 and ep-
othilone PKS,9 are reported to catalyze the macrocycliza-
tion of linear polyketides, although a comprehensive
analysis of the macrocycle forming activities of PKS TE
domains using unnatural types of polyketide chains has
never been investigated. Only the hydrolytic activities of
the PKS TE domains have been reported to date.10–12 In
the present paper, we describe the oligomerization and
macrocyclization activities of the vicenistatin PKS TE do-
main (VinTE) in the presence of several ω-hydroxy fatty
acid ethyl esters (Scheme 1). Although TE have been re-
ported to be active towards thioesters including N-acetyl-
H
Me
O
N
O
N
H
VinC
OH
O
vicenistatin
Scheme 1 Function of VinTE in vicenistatin biosynthesis. VinTE:
thioesterase (TE) domain of VinP4 polyketide synthase (PKS), VinC:
vicenisaminyltransferase, ACP: acyl carrier protein.
The VinTE protein was prepared according to a modified
method from a previous report.6 The N-terminal His-
tagged VinTE was expressed in Escherichia coli and pu-
rified with Ni-affinity chromatography. After removal of
imidazole and concentration by ultrafiltration, VinTE was
found to have the same activity as the native protein. To
examine the macrocyclization activity of VinTE, ω-hy-
droxy fatty acid ethyl esters in DMSO (final 1–3 mM and
10% DMSO) were mixed with VinTE in 100 mM sodium
phosphate buffer (pH 7.0), and the mixtures were incubat-
ed at 28 °C for 24 hours. Enzymatic reaction products
were extracted with excess amounts of chloroform, and
the extract was purified by silica gel chromatography to
isolate the reaction products (see details in the Supporting
Information).
The shortest examined substrate, ethyl 6-hydroxyhexano-
ate (1), was polymerized and macrocyclized to afford cy-
clic trimer 12 and tetramer 13 in 49% and 10% yield,
respectively (Scheme 2). Ethyl 8-hydroxyoctanoate (2)
and ethyl (E)-8-hydroxy-2-octenoate (3) were converted
into the corresponding cyclic dimers 14 and 16 and tri-
mers 15 and 17. In the case of the C-8 substrates 2 and 3,
significant amounts of the linear polyesters were also iso-
SYNLETT 2012, 23, 1843–1846
Advanced online publication: 21.06.2012
0
9
3
6
-
5
2
1
4
1
4
3
7
-
2
0
9
6
DOI: 10.1055/s-0031-1290385; Art ID: ST-2012-Y0097-C
© Georg Thieme Verlag Stuttgart · New York

1844
F. Kudo et al.
LETTER
O
O
O
O
O
VinTEa
O
O
O
O
21
28
27
+
HO
O
O
OEt
O
1
O
O
O
12 (49%)
13 (10%)
O
O
O
O
O
VinTEa
O
O
linear oligomers
(ca. 30%)
18
O
+
+
+
+
HO
O
OEt
OEt
O
O
2
3
14 (26%)
O
15 (7%)
O
O
O
O
VinTEa
linear oligomers
(ca. 15%)
O
O
18
27
HO
O
O
O
16 (37%)
O
17 (trace)
O
O
OEt OH
VinTEa
O
O
O
O
26
+
HO
O
O
O
OEt
OEt
4
5
O
O
O
18 (52%)
19 (32%)
OEt OH
VinTEa
O
O
+
26
O
HO
O
20 (51%)
21 (30%)
HO
O
OH
O
VinTEb
O
26
O
HO
SNAC
O
OH
6
22 (43%)
O
OH
O
O
VinTEa
HO
OEt
OH
17
7
OH
OH
23 (53%)
VinTEa
O
O
19
O
HO
OEt
OEt
8
9
O
24 (83%)
H
HO
N
OH
VinTEc
O
H2N
20
25 (51%)
VinTEa
VinTEa
O
O
no cyclic product
H2N
OEt
OEt
10
11
no cyclic product
H2N
Scheme 2 VinTE reaction with ω-hydroxy fatty acid ethyl esters. a 60 μM of VinTE, 1–3 mM of substrate, 28 °C, 24 h; b 300 μM of VinTE, 7
mM of substrate, 28 °C, 24 h. c 130 μM of VinTE, 1.25 mM of substrate, 28 °C, 40 min.6
Synlett 2012, 23, 1843–1846
© Georg Thieme Verlag Stuttgart · New York

LETTER
Oligomerization and Macrocyclization by VinTE
1845
lated. Ethyl 12-hydroxydodecanoate (4) and ethyl (Z)-12- linear ester. The formed linear dimer is then released from
hydroxy-9-dodecenoate (5) were converted into respec- the active site and is recognized again by VinTE, and the
tive linear dimers 19 and 21 and cyclic dimers 18 and 20. subsequent intramolecular nucleophilic attack gives the
The expected monocyclic compound from 5, the Yuzu cyclic dimer. When an additional ω-hydroxy ester enters
lactone a volatile component of Yuzu fruit,14 was not the active site in the presence of a dimeric linear ester, the
formed at all. The N-acetylcysteamine thioester (6), which subsequent polymerization occurs to form a linear trimer.
is a synthetic intermediate of the macrolide antibiotic FD- Then, the longer ω-hydroxy fatty acid ester can no longer
892,15 was converted into cyclic dimer 22 in 43% yield. be cyclized because of the space limitation of the active
Finally, the aleuritic acid ethyl ester (7) and ethyl (Z)-18- site of VinTE. A structural model of VinTE (Figure S3 in
hydroxy-9-octadecenoate (8) were converted into mono- the Supporting Information) showed the appropriate size
cyclic molecules 23 and 24, respectively. The formed 19- of the hydrophobic pocket to recognize the lipophilic 20-
membered macrocycle 23 is a known musk compound in membered vicenilactam. Indeed, the well-conserved ami-
tobacco.16 As we previously reported, the ethyl ester of the no acid residues including the critical serine residue seem
natural secovicenilactam was efficiently cyclized by to comprise a catalytic triad. The enterobactin NRPS EntF
VinTE for 40 minutes.6 However, the ω-amino fatty acid TE domain21 and gramicidin S NRPS TE GrsB TE
ethyl esters 10 and 11 were not cyclized at all.
domain22 are known to catalyze both oligomerization and
cyclization of elongated polypeptides. The TE domain of
a hybrid NRPS/PKS was also hypothesized to be respon-
sible for both the dimerization and cyclization in the bio-
synthesis of disorazole.23 Therefore, the observed
oligomerization–macrocyclization activity of the PKS TE
domain VinTE appeared to be a general feature in
PKS/NRPS assembly lines. Cyclic polyketides such as
tartolon D24 could be constructed by the use of thioester-
ase chemistry in a producer strain.
Consequently, it was found that VinTE catalyzes the
oligomerization and macrocyclization of ω-hydroxy fatty
acid ethyl esters. The ring sizes of the formed macrocycles
were limited to 17–28-membered rings (Scheme 2), pre-
sumably due to the cavity size of the active site pocket of
VinTE. The structural model of VinTE (Figure S3 in the
Supporting Information) based on the crystal structure of
the picromycin PKS-TE domain (PicTE or PikTE)17–19 re-
vealed that VinTE has a slightly larger cavity against 20-
membered vicenilactam. Therefore, VinTE appear to ac- To investigate the generality of the macrocyclization ac-
cept slightly larger ω-hydroxy fatty acid polyesters for tivity of PKS TEs with ω-hydroxy fatty acid ethyl esters,
macrocyclization. On the other hand, with the longer we examined the catalytic activity of HlsTE in halstocta-
length of ω-hydroxy fatty acid esters that are not accom- cosanolide PKS25 derived from the same producer as vice-
modated in the active site of enzyme, VinTE just catalyzes nistatin and GfsTE in FD-891 PKS26 derived from
the polymerization to afford linear polymers. It should be Streptomyces graminofaciens. However, no macrocyclic
noted that, in the vicenistatin biosynthesis, the N-alanyl- products were detected in the presence of HlsTE or
secovicenilactam biosynthetic intermediate must be de- GfsTE. Thus, the oligomerization–macrocyclization ac-
protected by a peptidase VinJ to secovicenilactam thioes- tivity against ω-hydroxy fatty acid esters seemed to be a
ter before macrocyclization by VinTE.20
specific property of VinTE, probably due to the high hy-
drophobicity in its active site.
To understand the reaction mechanism of oligomerization
and macrocyclization catalyzed by VinTE, the isolated Macrocyclization is a featured transformation in organic
linear dimer 21 was reacted again with VinTE (Scheme synthesis, because numerous substances including bioac-
3). As a result, the expected cyclized dimer 20 was pro- tive natural products often possess macrocyclic structures.
duced in high yield. Furthermore, the presumed active site Therefore, many synthetic methods27 including macrolac-
amino acid residue serine 136 (S136) of VinTE (Figures tonization,28 Prins-type reaction,29 and ring-closing me-
S3 and S4 in the Supporting Information) was mutated to tathesis,30 have been developed. In addition, enzymatic
alanine resulting in no activity. Therefore, S136 of VinTE polymerization and macrocyclization of carboxylic acids,
was found to be a critical catalytic residue. Considering esters, and monomeric lactones have been developed us-
these results, the dimerization and macrocyclization ing lipase.31 Thus, chemoenzymatic approach32 with the
mechanism is proposed as follows. The active site of reported VinTE should provide a new useful method for
VinTE accommodates two ω-hydroxy fatty acid ester sub- synthetic biological chemistry. If one can control the
strates, and the dimerization is triggered by nucleophilic length of polymers and ring sizes in the VinTE-mediated
attack of the hydroxy group of the active site S136 to one reactions, VinTE would become a powerful enzymatic
substrate and the subsequent nucleophilic attack from the tool.
hydroxy group of another substrate leading to a dimeric
OEt OH
O
O
VinTE
O
VinTE
O
O
x 2
O
HO
OEt
O
O
Scheme 3 Mechanism of VinTE reaction
© Georg Thieme Verlag Stuttgart · New York
Synlett 2012, 23, 1843–1846

1846
F. Kudo et al.
LETTER
(14) Rodefeld, L.; Tochtermann, W. Tetrahedron 1998, 54, 5893.
(15) Eguchi, T.; Yamamoto, K.; Mizoue, K.; Kakinuma, K.
J. Antibiot. 2004, 57, 156.
Acknowledgment
This work was supported in part by Grants-in-Aid for Scientific Re-
search on Innovative Areas from the Ministry of Education, Cultu-
re, Sports, Science and Technology (MEXT) and the Naito
Foundation.
(16) Demole, E.; Enggist, P. Helv. Chim. Acta 1978, 61, 2318.
(17) Tsai, S. C.; Miercke, L. J.; Krucinski, J.; Gokhale, R.; Chen,
J. C.; Foster, P. G.; Cane, D. E.; Khosla, C.; Stroud, R. M.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 14808.
(18) Giraldes, J. W.; Akey, D. L.; Kittendorf, J. D.; Sherman, D.
H.; Smith, J. L.; Fecik, R. A. Nat. Chem. Biol. 2006, 2, 531.
(19) Akey, D. L.; Kittendorf, J. D.; Giraldes, J. W.; Fecik, R. A.;
Sherman, D. H.; Smith, J. L. Nat. Chem. Biol. 2006, 2, 537.
(20) Shinohara, Y.; Kudo, F.; Eguchi, T. J. Am. Chem. Soc. 2011,
133, 18134.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synlett.SnoIufproig
m
iotSrat
ungIifoop
r
t
References
(1) Kopp, F.; Marahiel, M. A. Nat. Prod. Rep. 2007, 24, 735.
(2) Kohli, R. M.; Walsh, C. T. Chem. Commun. 2003, 297.
(3) Kohli, R. M.; Trauger, J. W.; Schwarzer, D.; Marahiel,
M. A.; Walsh, C. T. Biochemistry 2001, 40, 7099.
(4) Kohli, R. M.; Walsh, C. T.; Burkart, M. D. Nature (London)
2002, 418, 658.
(21) Shaw-Reid, C. A.; Kelleher, N. L.; Losey, H. C.; Gehring,
A. M.; Berg, C.; Walsh, C. T. Chem. Biol. 1999, 6, 385.
(22) Hoyer, K. M.; Mahlert, C.; Marahiel, M. A. Chem. Biol.
2007, 14, 13.
(23) Carvalho, R.; Reid, R.; Viswanathan, N.; Gramajo, H.;
Julien, B. Gene 2005, 359, 91.
(5) Kohli, R. M.; Takagi, J.; Walsh, C. T. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 1247.
(6) Kudo, F.; Kitayama, T.; Kakinuma, K.; Eguchi, T.
Tetrahedron Lett. 2006, 47, 1529.
(24) Perez, M.; Crespo, C.; Schleissner, C.; Rodriguez, P.;
Zuniga, P.; Reyes, F. J. Nat. Prod. 2009, 72, 2192.
(25) Tohyama, S.; Kakinuma, K.; Eguchi, T. J. Antibiot. 2006,
59, 44.
(7) Aldrich, C. C.; Venkatraman, L.; Sherman, D. H.; Fecik, R.
A. J. Am. Chem. Soc. 2005, 127, 8910.
(26) Kudo, F.; Motegi, A.; Mizoue, K.; Eguchi, T.
ChemBioChem 2010, 11, 1574.
(8) He, W.; Wu, J.; Khosla, C.; Cane, D. E. Bioorg. Med. Chem.
Lett. 2006, 16, 391.
(27) Madsen, C. M.; Clausen, M. H. Eur. J. Org. Chem. 2011,
3107.
(9) Boddy, C. N.; Schneider, T. L.; Hotta, K.; Walsh, C. T.;
Khosla, C. J. Am. Chem. Soc. 2003, 125, 3428.
(10) Gokhale, R. S.; Hunziker, D.; Cane, D. E.; Khosla, C. Chem.
Biol. 1999, 6, 117.
(28) Parenty, A.; Moreau, X.; Campagne, J. M. Chem. Rev. 2006,
106, 911.
(29) Crane, E. A.; Scheidt, K. A. Angew. Chem. Int. Ed. 2010, 49,
8316.
(11) Lu, H.; Tsai, S. C.; Khosla, C.; Cane, D. E. Biochemistry
2002, 41, 12590.
(12) Wang, M.; Opare, P.; Boddy, C. N. Bioorg. Med. Chem. Lett.
2009, 19, 1413.
(13) Grunewald, J.; Sieber, S. A.; Marahiel, M. A. Biochemistry
2004, 43, 2915.
(30) Hassan, H. M. A. Chem. Commun. 2010, 46, 9100.
(31) Dong, H.; Wang, H.-D.; Cao, S.-G.; Shen, J.-C. Biotechnol.
Lett. 1998, 20, 905.
(32) Mortison, J. D.; Sherman, D. H. J. Org. Chem. 2010, 75,
7041.
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