Epoxide hydrolase Lsd19 for polyether formation in the biosynthesis of lasalocid A: Direct experimental evidence on polyene-polyepoxide hypothesis in polyether biosynthesis

Article abstract of DOI:10.1021/ja8040543

Polyether metabolites are an important class of natural products. Although their biosynthesis, especially construction of polyether skeletons, attracted organic chemists for many years, no experimental data on the enzymatic polyether formation has been obtained. In this study, a putative epoxide hydrolase gene lsd19 found on the biosynthetic gene cluster of an ionophore polyether lasalocid was cloned and successfully overexpressed in Escherichia coli. Using the purified Lsd19, a proposed substrate, bisepoxyprelasalocid, and its synthesized analogue were successfully converted into lasalocid A and its derivative via a 6-endo-tet cyclization mode. On the other hand, treatment of the bisepoxide with trichloroacetic acid gave isolasalocid A via a 5-exo-tet cyclization mode. Therefore, the enzymatic conversion observed in this study unambiguously showed that the bisepoxyprelasalocid is an intermediate of the lasalocid biosynthesis and that Lsd19 catalyzes the sequential cyclic ether formations involving an energetically disfavored 6-endo-tet cyclization. This is the first example of the enzymatic epoxide-opening reactions leading to a polyether natural product. Copyright

Full text of DOI:10.1021/ja8040543

Published on Web 08/19/2008
Epoxide Hydrolase Lsd19 for Polyether Formation in the Biosynthesis of
Lasalocid A: Direct Experimental Evidence on Polyene-Polyepoxide
Hypothesis in Polyether Biosynthesis
Yoshihiro Shichijo,† Akira Migita,† Hiroki Oguri,*,† Mami Watanabe,† Tetsuo Tokiwano,†,
Kenji Watanabe,‡,§ and Hideaki Oikawa*,†
DiVision of Chemistry, Graduate School of Science, Hokkaido UniVersity, Sapporo 060-0810, Japan and
Department of Pharmacology and Pharmaceutical Sciences, UniVersity of Southern California,
Los Angeles, California 90033
Received June 4, 2008; E-mail: hoik@sci.hokudai.ac.jp; oguri@sci.hokudai.ac.jp
Lasalocid A1 (1) isolated from Streptomyces lasaliensis is one
Scheme 1. Proposed Biosynthetic Pathways of Representative
Polyether Antibiotics
of the important ionophore antibiotics among commercially avail-
able anticoccidial agents. Feeding experiments suggested that 1 is
biosynthesized via a dodecaketide.2,3 Considering coproduction of
isolasalosid A (2) along with 1 in S. lasaliensis, Westley and co-
workers proposed that the stereoselective epoxidation of a dode-
caketide precursor prelasalocid (3) and the sequential ring openings
of the resultant bisepoxide 4 would afford the ether ring system of
1 as shown in Scheme 1A.4 In the biosynthetic studies on another
important polyether ionophore monensin (6), this insightful hy-
pothesis was supported by extensive incorporation experiments with
[1-13C,1-18O]-short chain fatty acids and 18O2, indicating that
terminal three oxygen atoms are derived from the corresponding
epoxidation of the triene precursor 7 as shown in Scheme 1B.5
These data led to the Cane-Celmer-Westley unified hypothesis
of ionophore polyether biosynthesis in actinomycetes.6 After this
proposal, several groups attempted to incorporate advanced inter-
Scheme 2a
mediates such as 7; however, all efforts to prove this attractive
hypothesis failed.7 In 2001, the gene cluster of monensin was
indentified,8,9 and several gene disruption experiments on epoxidase
(MonCI) and hydrolases (MonBI, BII) established that these three
enzymes are responsible for the conversion of all-E triene 7 into 6
via epoxidation and cyclization.10,11 Yet, a detailed mechanism of
a Reagents and conditions: (a) A, OxoneR, K2CO3, Bu4NHSO4,
CH3CN-CH2(OCH3)2-H2O, 0 °C, 2 h, 10 (52%), diastereomer (17%); (b)
TBAF (0.4 equiv), THF, rt, 30 min, 11 (24%), 10 (74%, recovery); (c) H2,
the enzymatic polyether formation is still unconfirmed. Herein, we
report the first enzymatic conversions of bisepoxides 4 and 11 into
lasalocid skeletons.
Pd(OH)2, MeOH, rt, 10 min, 98%; (d) H2, Pd(OH)2, MeOH/aq. NH4HCO3,
A total DNA library screening using a probe which was prepared
by PCR with degenerate primers designed for the ketosynthase
domains of polyketide synthase allowed us to identify the gene
cluster responsible for lasalocid biosynthesis.12 Among the lasalocid
biosynthetic genes, lsd19, which showed significant homology to
the putative epoxide hydrolase genes monBI and monBII, was
assumed to be responsible for the construction of the polyether
skeleton.11 The key gene, lsd19, was cloned and successfully
expressed in Escherichia coli BL21(DE3) by the use of the
expression plasmid pKW620.13 Cell lysate containing Lsd19
expressed as an N-terminal hexa-His-tagged protein was purified
using Ni-NTA column chromatography to give Lsd19 at >90%
purity as measured by SDS-PAGE analysis with a yield of
approximately 2 mg/L of culture.
pH 7.8, 4 (3%), 11 (90% recovery).
synthesize bisepoxyprelasalocid 4, a proposed substrate of Lsd19,
epoxidation of suitably protected prelasalocid 9 with Shi’s catalyst15
proceeded in a stereoselective manner to give 10 and its diastere-
omer in a 3:1 ratio as shown in Scheme 2.16 After deprotection of
the TES ether, hydrogenolysis under slightly basic conditions
effected removal of the benzyl groups to provide desired 4.
However, the bisepoxide 4 is prone to undergo epoxide opening
presumably due to acidic functionalities on the aromatic ring,
thereby forming significant amounts of monocyclic ether 5 and
isolasalosid A (2) having adjacent ether rings during prolonged
exposure to the hydrogenolysis conditions and purification. Thus,
the mixture of bisepoxides, 4 and 11, was subjected immediately
to the enzymatic reaction with Lsd19.
LC-MS analysis of bisepoxide 4 showed that the substrate
contained nonenzymatic cyclization products 2 and 5. This was
confirmed by the fact that treatment of the substrate mixture with
trichloroacetic acid gave 5-exo-tet cyclization product 2 as a single
diastereomer (Figure 1A) in accordance with Baldwin’s rules. On
the other hand, incubation of the same substrate mixture with the
Recently, we have reported the stereocontrolled synthesis of
prelasalocid (3), a plausible biosynthetic precursor of 1.14 To
† Hokkaido University.
‡ University of Southern California.
§ Current address: Division of Chemistry, Graduate School of Science, Hokkaido
University, Sapporo 060-0810, Japan.
Current address: Department of Biotechnology, Faculty of Bioresource
Sciences, Akita Prefectural University, Akita 010-0195, Japan.
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2008 American Chemical Society

C O M M U N I C A T I O N S
in intramolecular cyclic ether formation. Currently, studies on
substrate specificity and the reaction mechanism of Lsd19 are in
progress.
Acknowledgment. We are grateful to Mr. Yusuke Matsuura in
our laboratory for measuring spectral data and to Kaken Pharma-
ceutical Co. Ltd. for supplying generous amounts of natural
lasalocid A. This work was supported by the Mitsubishi Foundation
and the Grants-in-Aid for Scientific Research [(A)17208010 to H.
Oikawa and 17710175 to H. Oguri] from the Japan Society for the
Promotion of Science (JSPS). A fellowship to A.M. from JSPS is
gratefully acknowledged.
Figure 1. HPLC-MS analysis of sequential ether formation products either
with acid or with Lsd19 (ESI positive mode). (A) Bisepoxyprelasalocid
(4): (i) mixture containing substrate 4 and cyclization products 2 and 5; (ii)
authentic samples of the products 1 and 2; (iii) reaction of 4 with
trichloroacetic acid; (iv) reaction of 4 with Lsd19. (B) Bisepoxide 11: (i)
mixture containing substrate 11 and monocyclic ether (denoted by *); (ii)
authentic samples of the products 1a and 2a; (iii) reaction with trichloroacetic
acid; (iv) reaction with Lsd19. Assays and HPLC conditions are available
in the Supporting Information.
Supporting Information Available: Experimental procedures and
characterization of compounds 1a, 2, 2a, and 9-11. This material is
available free of charge via the Internet at http://pubs.ac.jp.
References
purified Lsd19 predominantly afforded a 6-endo-tet cyclization
product which was identical to lasalocid A (1) (Figure 1A).17
Therefore, the enzymatic conversion of 4 into 1 unambiguously
showed that bisepoxide 4 is an intermediate for lasalocid biosyn-
thesis and that Lsd19 catalyzes sequential cyclic ether formation
involving an energetically disfavored 6-endo-tet cyclization. This
is the first example of enzymatic epoxide-opening reactions leading
to a polyether natural product. In addition, detection of the
monocyclic ether 5 and its conversion to 1 indicated that bicyclic
ether formation occurs in a stepwise manner. To confirm the
enzymatic activity of Lsd19, enzymatic reaction with the substrate
analogue 11 was employed. Incubation of 11 with Lsd19 afforded
6-endo-tet cyclization product 1a as a single diastereomer, while
treatment of 11 with trichloroacetic acid gave 5-exo-tet cyclization
product 2a (Figure 1B). Thus, the experimental results clearly
showed that Lsd19 is responsible for the desired polyether formation
reaction to give the lasalocid skeleton.
In the biosynthesis of monensin and nanchangmycin, it has been
proposed that epoxidation and cyclization occurred when intermedi-
ates were bound to a polyketide synthase (PKS).18,19 Our experi-
mental results indicate that in the case of lasalocid the polyether
formation most likely occurs after the polyketide chain is cleaved
from PKS,12 but further study is needed to exclude the possibility
that a full-length polyketide bound to ACP is a mandatory substrate.
An intramolecular ether formation of hydroxyepoxide has posed
an intriguing problem in organic chemistry. In an effort to construct
ladder polyethers found in marine dinoflagelate toxins, such as
brevetoxin and cigatoxin, a number of synthetic protocols to
overcome the energetically disfavored 6-endo-tet cyclization have
been developed.20,21 Recently, Jamison and his co-workers have
reported that 6-endo cyclization proceeded nonenzymatically in an
aqueous medium without any acid or base catalysis.22 Although
this proposal is attractive for synthesizing the fused cyclic ether
system of the ladder polyethers, it seems unlikely to explain the
universal biosynthetic mechanisms of marine polyether natural
products. Alternatively, a catalytic antibody catalyzing the disfa-
vored intramolecular cyclization of simple hydroxyepoxide has been
created.23 A structural study of this antibody provided useful
information on its catalytic mechanism.24 However, to date, little
is known about the enzyme catalysis on sequential cyclic ether
formation. Therefore, we believe that Lsd19 can be regarded as a
model enzyme for studying multiple catalysis and regioselectivity
(1) Westley, J. W.; Evans, R. H., Jr.; Williams, T.; Stempel, A. Chem. Commun.
1970, 71.
(2) Westley, J. W.; Evans, R. H.; Harvey, G.; Pitcher, R. G.; Pruess, D. L. J.
Antibiot. 1974, 27, 288–297.
(3) Hutchinson, C. R.; Sherman, M. M.; Vederas, J. C.; Nakashima, T. T. J. Am.
Chem. Soc. 1981, 103, 5953–5956.
(4) Westley, J. W.; Blount, J. F.; Evans, R. H., Jr.; Stempel, A.; Berger, J. J.
Antibiot. 1974, 27, 597–604.
(5) Cane, D. E.; Liang, T. C.; Hasler, H. J. Am. Chem. Soc. 1981, 103, 5962–
5965.
(6) Cane, D. E.; Celmer, W. D.; Westley, J. W. J. Am. Chem. Soc. 1983, 105,
3594–3600.
(7) Holmes, D. S.; Sherringham; J. A.; Dyer, U. C.; Russell, S. T.; Robinson,
J. A. HelV. Chim. Acta 1990, 73, 239–259, and references cited therein.
(8) Leadlay, P. F.; Staunton, J.; Oliynyk, M.; Bisang, C.; Cortes, J.; Frost, E.;
Hughes-Thomas, Z. A.; Jones, M. A.; Kendrew, S. G.; Lester, J. B.; Long,
P. F.; McArthur, H. A. I.; McCormick, E. L.; Oliynyk, Z.; Stark, C. B. W.;
Wilkinson, C. J. J. Ind. Microbiol. Biotechnol. 2001, 27, 360–367.
(9) Oliynyk, M.; Stark, C. B. W.; Bhatt, A.; Jones, M. A.; Hughes-Thomas,
Z. A.; Wilkinson, C.; Oliynyk, Z.; Demydchuk, Y.; Staunton, J.; Leadlay,
P. F. Mol. Microbiol. 2003, 49, 1179–1190.
(10) Bhatt, A.; Stark, C. B.; Harvey, B. M.; Gallimore, A. R.; Demydchuk,
Y. A.; Spencer, J. B.; Staunton, J.; Leadlay, P. F. Angew. Chem., Int. Ed.
2005, 44, 7075–7078.
(11) Gallimore, A. R.; Stark, C. B.; Bhatt, A.; Harvey, B. M.; Demydchuk, Y.;
Bolanos-Garcia, V.; Fowler, D. J.; Staunton, J.; Leadlay, P. F.; Spencer,
J. B. Chem. Biol. 2006, 13, 453–460.
(12) Genetic information of lasalocid and discussion of the timing of cleavage
of full-length polyketide intermediate from PKS: Migita, A.; Watanabe,
M.; Hirose, Y.; Watanabe, K.; Tokiwano, T.; Kinashi, H.; Oikawa, H.
Submitted. The DNA sequence of lsd19 has been deposited with DDBJ
(Accession no.: AB438996).
(13) Experimental details on cloning and overexpression of lsd19 are described
in the Supporting Information.
(14) Migita, A.; Shichijo, Y.; Oguri, H.; Watanabe, M.; Tokiwano, T.; Oikawa,
H. Tetrahedron Lett. 2008, 49, 1021–1025.
(15) Wang, Z. X.; Tu, Y.; Frohn, M.; Zhang, J. R.; Shi, Y. J. Am. Chem. Soc.
1997, 119, 11224–11235.
(16) The structure of bisepoxide 11 was determined by its conversion to
isolasalocid A (2) and isolasalocid ketone; see Supporting Information.
(17) Incubation of 5-exo product 2 with Lsd19 under the same assay conditions
did not produce 1 at all, which excludes possibilities that Lsd19 mediates
a rearrangement of the terminal tetrahydrofuran into the tetrahydropyran.
(18) Harvey, B. M.; Hong, H.; Jones, M. A.; Hughes-Thomas, Z. A.; Goss,
R. M.; Heathcote, M. L.; Bolanos-Garcia, V. M.; Kroutil, W.; Staunton,
J.; Leadlay, P. F.; Spencer, J. B. ChemBioChem 2006, 7, 1435–1442.
(19) (a) Liu, T.; You, D.; Valenzano, C.; Sun, Y.; Li, J.; Yu, Q.; Zhou, X.;
Cane, D. E.; Deng, Z. Chem. Biol. 2006, 13, 945–55. (b) Liu, T.; Lin, X.;
Zhou, X.; Deng, Z.; Cane, D. E. Chem. Biol. 2008, 15, 449–458.
(20) (a) Nicolaou, K. C.; Prasad, C. V. C.; Somers, P. K.; Hwang, C.-K. J. Am.
Chem. Soc. 1989, 111, 5330–5334. (b) Fujiwara, K.; Murai, A. Bull. Chem.
Soc. Jpn. 2004, 77, 2129–2146.
(21) McDonald, F. E.; Tong, R. B.; Valentine, J. C.; Bravo, F. Pure Appl. Chem.
2007, 79, 281–291.
(22) Vilotijevic, I.; Jamison, T. F. Science 2007, 317, 1189–1192.
(23) Janda, K. D.; Shevlin, C. G.; Lerner, R. A. Science 1993, 259, 490–493.
(24) Gruber, K.; Zhou, B.; Houk, K. N.; Lerner, R. A.; Shevlin, C. G.; Wilson,
I. A. Biochemistry 1999, 38, 7062–7074.
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