Intriguing substrate tolerance of epoxide hydrolase Lsd19 involved in biosynthesis of the ionophore antibiotic lasalocid A

Article abstract of DOI:10.1021/ol100541e

Recently, we reported that the epoxide hydrolase Lsd19, the first enzyme shown to catalyze epoxide-opening cascades, can catalyze the conversion of a putative bisepoxide intermediate to polyether antibiotic lasalocid, which involves energetically disfavored 6-endo-tet cyclization of the epoxy alcohol. Here, we examined the substrate tolerance of Lsd19. Lsd19 accepts various substrate analogues differing in the left segment of lasalocid and epoxide stereochemistry to afford either THF-THP or THF-THF products with excellent regioselectivity.

Full text of DOI:10.1021/ol100541e

ORGANIC
LETTERS
2010
Vol. 12, No. 10
2226-2229
Intriguing Substrate Tolerance of
Epoxide Hydrolase Lsd19 Involved in
Biosynthesis of the Ionophore Antibiotic
Lasalocid A
Yusuke Matsuura, Yoshihiro Shichijo, Atsushi Minami, 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
hoik@sci.hokudai.ac.jp
Received March 5, 2010
ABSTRACT
Recently, we reported that the epoxide hydrolase Lsd19, the first enzyme shown to catalyze epoxide-opening cascades, can catalyze the
conversion of a putative bisepoxide intermediate to polyether antibiotic lasalocid, which involves energetically disfavored 6-endo-tet cyclization
of the epoxy alcohol. Here, we examined the substrate tolerance of Lsd19. Lsd19 accepts various substrate analogues differing in the left
segment of lasalocid and epoxide stereochemistry to afford either THF-THP or THF-THF products with excellent regioselectivity.
Naturally occurring polyether metabolites include ionophore
antibiotics,1 marine dinoflagellate toxins,2 marine triterpenes,3
and annonaceous antitumor acetogenins.4 It has been pro-
posed that these polyether skeletons are constructed by
nucleophilic epoxide-opening cascades of the corresponding
polyepoxides after installing a number of stereogenic centers
on the simple polyolefins in enzymatic epoxidation.5,6 This
simple yet versatile strategy for the biosynthesis of complex
polyether metabolites prompted chemists to synthesize iono-
phore antibiotics and marine dinoflagellate toxins using
epoxide-opening cascades. The successful biomimetic syn-
thesis of various polyether systems supports these biosyn-
thetic proposals.7 An intrinsic problem with this strategy is
regioselective opening of polyepoxides. According to Bald-
win’s rules, the epoxide-opening reaction of hydroxyepoxides
usually favors the formation of smaller cyclic ethers. Thus,
† Present address: Department of Biotechnology, Faculty of Bioresource
Sciences, Akita Prefectural University, Akita 010-0195, Japan.
‡ Present address: School of Pharmaceutical Sciences, University of
Shizuoka, Shizuoka, 422-8526, Japan.
(1) Dutton, C. J.; Banks, B. J.; Cooper, C. B. Nat. Prod. Rep. 1995, 12,
165–181.
(2) Murata, M.; Yasumoto, T. Chem. ReV. 1993, 93, 1897–1909.
(3) Fernandez, J. J.; Souto, M. L.; Norte, M. Nat. Prod. Rep. 2000, 17,
235–246.
(5) Cane, D. E.; Celmer, W. D.; Westley, J. W. J. Am. Chem. Soc. 1983,
105, 3594–3600.
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(7) Vilotijevic, I.; Jamison, T. F. Angew. Chem., Int. Ed. 2009, 48, 5250–
5281.
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Estornell, E.; Cortes, D. Nat. Prod. Rep. 2005, 22, 269–303.
10.1021/ol100541e  2010 American Chemical Society
Published on Web 04/15/2010

in the case of competitive formation of THF (5-exo-tet) and
THP (6-endo-tet), 6-endo cyclization to give THP is an
energetically disfavored process. Biosynthetic anti-Baldwin
cyclization has been proposed to involve a specific enzyme
that stabilizes the disfavored transition state.6
Starting from the synthetic intermediates of prelasalocid
reported previously,14 the substrate analogues 5-7 and 13a
were synthesized under essentially the same conditions as
bisepoxides 3 and 3a (Scheme 2).13 First, incubation of the
Among natural polyethers, the biosynthesis of ionophore
antibiotics has been studied extensively. In 1983, Cane,
Celmer, and Westley proposed a unified biogenetic scheme
for ionophore antibiotics based on the results of a series of
feeding experiments.5 The CCW unified biogenesis has been
supported by identification and functional analysis of bio-
synthetic genes.8-11 Recently, we reported the identification
of the lasalocid biosynthetic gene cluster12 and showed that
the epoxide hydrolase Lsd19 can catalyze conversion of a
putative bisepoxide intermediate 3 to lasalocid A (1) via 4,
which involves energetically disfavored 6-endo-tet cycliza-
tion of the epoxyalcohol (Scheme 1).13 On the other hand,
Scheme 2.
Enzymatic Reaction with Epoxide Hydrolase Lsd19a
Scheme 1. Enzymatic Reaction with Epoxide Hydrolase Lsd19
Affording Polyether Antibiotic Lasalocid A (1)
acid treatment of 3 gave isolasalocid A (2) exclusively. The
first direct experimental evidence sets the stage for investi-
gating enzymatic polyether formation in vitro. In our previous
study, we showed that Lsd19 is capable of converting
bisbenzyl analog 3a to 1a.13 This relaxed substrate specificity
suggests that Lsd19 is an ideal model enzyme for mechanistic
studies of the intriguing epoxide-opening cascade, using
various epoxide analogues. Here, we describe the relatively
broad substrate tolerance of Lsd19.
a (A) Substrates with different left-hand structures. (B) Substrates with
different epoxide stereochemistry.
C12-C24-bisepoxide 5 lacking a salicylate moiety with
purified Lsd19 was carried out. LC-MS analysis of the
reaction mixture indicated that enzymatic reaction exclusively
afforded THF-THP (lasalocid-type) product 8,13 whereas
treatment of 5 with trichloroacetic acid (TCA) gave the
energetically favored THF-THF (isolasalocid-type) 1013 as
a single product. Simple C15-C24-bisepoxide 6 and bise-
poxide 13a with an oxazolidinone were also converted into
the corresponding THF-THP products 9 and 15a, respec-
tively. On the other hand, C19-C24-monoepoxide 7 did not
give any reaction product (data not shown).
(8) 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
.
(9) Sun, Y. H.; Zhou, X. F.; Dong, H.; Tu, G. Q.; Wang, M.; Wang,
B. F.; Deng, Z. X. Chem. Biol. 2003, 10, 431–441
.
(10) Harvey, B. M.; Mironenko, T.; Sun, Y.; Hong, H.; Deng, Z.;
Leadlay, P. F.; Weissman, K. J.; Haydock, S. F. Chem. Biol. 2007, 14,
703–714
.
(11) Bhatt, A.; Stark, C. B.; Harvey, B. M.; Gallimore, A. R.;
Considering the successful conversions of the truncated
analogues 5 and 6 without the left segment of the putative
intermediate bisepoxyprelasalocid 3, the salicylate moiety
is not essential for substrate recognition, and Lsd19 accepts
Demydchuk, Y. A.; Spencer, J. B.; Staunton, J.; Leadlay, P. F. Angew.
Chem., Int. Ed. Engl. 2005, 44, 7075–7078
.
(12) Migita, A.; Watanabe, M.; Hirose, Y.; Watanabe, K.; Tokiwano,
T.; Kinashi, H.; Oikawa, H. Biosci. Biotechnol. Biochem. 2009, 73, 169–
176. See also: Smith, L.; Hong, H.; Spencer, J. B.; Leadlay, P. F.
ChemBioChem 2008, 9, 2967–2975. In this paper, the authors identifed
Lsd19 as LasB.
(13) Shichijo, Y.; Migita, A.; Oguri, H.; Watanabe, M.; Tokiwano, T.;
Watanabe, K.; Oikawa, H. J. Am. Chem. Soc. 2008, 130, 12230–12231.
(14) Migita, A.; Shichijo, Y.; Oguri, H.; Watanabe, M.; Tokiwano, T.;
Oikawa, H. Tetrahedron Lett. 2008, 49, 1021–1025.
Org. Lett., Vol. 12, No. 10, 2010
2227

Figure 1. HPLC-MS analysis of the products in the sequential ether formation with Lsd19 (ESI positive mode). Lsd19 assays were
performed in a total volume of 50 µL in Tris-HCl buffer (20 mM, pH 7.4) with 0.1 µM Lsd19 at 30 °C for 3 h including control experiments
without Lsd19. The substrate concentration was fixed at 1 µM to complete the reaction. All enzymatic reactions were carried out on a
preparative scale to identify major products by 1H NMR measurement: (A) (i) bisepoxide 5, (ii) reaction with Lsd19, (iii) authentic THF-THF
product 10; (B) (i) bisepoxide 6, (ii) reaction with Lsd19, (iii) authentic THF-THF product11; (C) (i) bisepoxide 13a, (ii) reaction with
Lsd19, (iii) authentic THF-THF product14a; (D) (i) bisepoxide 13b, (ii) reaction with Lsd19, (iii) authentic THF-THF product14b; (E)
(i) bisepoxide 13c, (ii) reaction without Lsd19, (iii) reaction with Lsd19; (F) (i) bisepoxide 13d, (ii) reaction without Lsd19, (iii) reaction
with Lsd19. The asterisk (*) indicates the corresponding monocyclic ether.
a broad range of substrate analogues differing in the left
segment of lasalocid. Acceptance of the bisepoxide 13a
bearing an oxazolidinone at the left end supports this
hypothesis.
reaction (Scheme 2B).16 All enzymatic reactions were nearly
completed within 3 h. Using each of the four diastereomeric
bisepoxides 13a-d, enzymatic reactions with Lsd19 were
performed (Scheme 2B). LC-MS analysis of the reaction
products with bisepoxides 13a and 13b revealed formation
of the THF-THP products 15a and 15b, respectively. On
the other hand, the enzymatic reaction of 13c and 13d
showed the predominant formation of THF-THF products
14c and 14d, respectively (Figure 1C-F).16
The structures of THF-THF products 14a-d obtained
by acid treatment of the corresponding bisepoxide diaster-
eomers 13a-d were determined by extensive NMR analysis
including NOESY spectra as shown in Figure 2. In addition,
the structures of THF-THP products15a and 15b afforded
in the Lsd19-catalyzed reaction were confirmed by com-
To examine the influence of epoxide stereochemistry in
the Lsd19-catalyzed reaction, epoxide diastereomers 13a-d
were synthesized by Shi’s asymmetric epoxidation of 12.15
Use of D-ligand A gave a pair of products 13a and 13b as a
2.7:1 mixture, while use of enantiomeric L-ligand ent-A
afforded a different set of bisepoxides, 13c and 13d, as a
7.5:1 mixture (Scheme 2B). Expected exo-cyclization prod-
ucts 14a-d were synthesized by acid treatment of the
corresponding epoxides. The corresponding endo-products
15a and 15b were synthesized by a two-step ring-expansion
(15) Wang, Z. X.; Tu, Y.; Frohn, M.; Zhang, J. R.; Shi, Y. J. Am. Chem.
Soc. 1997, 119, 11224–11235.
(16) Hori, N.; Nagasawa, K.; Shimizu, T.; Nakata, T. Tetrahedron Lett.
1999, 40, 2145–2148.
2228
Org. Lett., Vol. 12, No. 10, 2010

bound intermediate is an actual substrate for epoxidation and
the subsequent cascade cyclization.18 These factors made it
difficult to perform functional analysis of the epoxide
hydrolases MonBI and MonBII. Our study described in this
paper indicates that truncated substrates having an epoxy
alcohol moiety can be used for examining enzymatic activity
due to the broad substrate specificity of epoxide hydrolases.
If this is general in epoxide hydrolases in polyether antibiotic
biosynthesis, use of simple substrates would significantly
facilitate mechanistic study.
In the biosynthesis of monensin and nigericin, two epoxide
hydrolases may be responsible for three epoxide-opening
reactions because only two hydrolase genes were found in
the gene clusters.10,18 This implies that the epoxide hydrolase
involved in polyether biosynthesis catalyzes more than a
single reaction, and thus, small numbers of epoxide hydro-
lases are responsible for cascade cyclization of polyepoxide
substrates. From this point of view, the substrate specificity
of epoxide hydrolase is an intriguing topic for the polyether
biosynthesis of not only ionophore antibiotics but also other
polyether metabolites including marine ladder toxins. The
study of the detailed reaction mechanism of Lsd19-catalyzed
epoxide-opening reactions are currently underway in our
laboratory.
Figure 2. Selected NOE data for the cyclization products.
parison of their NMR spectra with those of the ring-
expansion products16 of 14a and 14b.
These experimental results showed that in the first THF
formation Lsd19 accepted both 18R,19R-(natural) and 18S,19S-
epoxides (unnatural) and that in the second cyclization Lsd19
switched the reaction pathways: (1) THP formation for
(22R,23R)-epoxide (ꢀ-epoxide, natural) and (2) THF forma-
tion for (22S,23S)-epoxide (R-epoxide, unnatural). These
remarkable regioselectivities provided further insight into the
reaction mechanism of Lsd19-catalyzed polyether construc-
tion. As described in our paper concerning identification of
the lasalocid gene cluster,12 Lsd19 has two catalytic domains
which are likely active. The role of two domains may be a
key to solve these intriguing observations. We are currently
working on the functional analysis of a series of Lsd19
mutants.
Synthesizing complex polyepoxide substrates and handling
these rather reactive compounds are difficult and are the
bottleneck of studying enzymatic epoxide-opening cascade
in the natural polyether biosynthesis. In monensin biosyn-
thesis, there are several cadidates for putative intermediates
of polyether formation due to late-stage modifications such
as O-methylation and C26 hydroxylation of polyketide
backbone.17 In addition, it was proposed that the enzyme-
Acknowledgment. This work was supported by Grants-
in-Aid for Scientific Research [(B) 20310126 to H. Oikawa
and Young Scientists (B) 21710213 to A. Minami] from the
Japan Society for the Promotion of Science (JSPS) and partly
by the Global COE Program (Project No. B01: Catalysis as
the Basis for Innovation in Material Science) from Mon-
bukagakusho. A fellowship to A. Migita from JSPS is
gratefully acknowledged.
Supporting Information Available: Experimental pro-
cedures and characterization of compounds 5-7, 9, 11,
13a-d, 14a-d, and 15a,b. This material is available free
of charge via the Internet at http://pubs.acs.org.
OL100541E
(18) (a) 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. (b) 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.
(17) 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.
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