Available online at www.sciencedirect.
com
Biosynthetic machinery of ionophore polyether lasalocid:
enzymatic construction of polyether skeleton
Atsushi Minami1, Hiroki Oguri1, Kenji Watanabe2 and Hideaki Oikawa1
Diversity of natural polycyclic polyethers originated from very
simple yet versatile strategy consisting of epoxidation of linear
polyene followed by epoxide opening cascade. To understand
two-step enzymatic transformations at molecular basis, a flavin
containing monooxygenase (EPX) Lsd18 and an epoxide
hydrolase (EH) Lsd19 were selected as model enzymes for
extensive investigation on substrate specificity, catalytic
mechanism, cofactor requirement and crystal structure. This
pioneering study on prototypical lasalocid EPX and EH
provides insight into detailed mechanism of ionophore
polyether assembly machinery and clarified remaining issues
for polyether biosynthesis.
Addresses
1
Division of Chemistry, Graduate School of Science, Hokkaido
University, Sapporo 060-0810, Japan
2
University of Shizuoka, School of Pharmaceutical Sciences, Shizuoka
422-8526, Japan
Corresponding author: Oikawa, Hideaki (hoik@sci.hokudai.ac.jp)
Current Opinion in Chemical Biology 2013, 17:555–561
This review comes from a themed issue on Mechanisms
Edited by Hung-wen Liu and Tadhg Begley
For a complete overview see the Issue and the Editorial
Available online 21st June 2013
1367-5931/$ – see front matter, # 2013 Elsevier Ltd. All rights
reserved.
http://dx.doi.org/10.1016/j.cbpa.2013.06.004
Introduction
Natural polycyclic polyethers such as ionophore poly-
ethers, ladder polyethers, antitumor acetogenins and
polycyclic oxasqualenoids are one of the structurally
unique classes of secondary metabolites (Figure 1).
Among these metabolites, ionophore polyethers [1] are
widely used as an anticoccidial drug in poultry and are fed
to ruminants to enhance feed efficiency [2]. They also act
as an anticancer, antimalarial and anti-human immuno-
deficiency virus agent [3–5]. The wide range of biological
activities depends on the structural diversity, basically
dictated by the number of ether rings and/or the manner
of ring closure. Following initial Westley’s speculation
[6], in 1983, Cane, Celmer and Westley proposed a simple
yet versatile biosynthetic model (CCW model) to con-
struct polycyclic polyether skeleton on the basis of the
labeling patterns and stereochemical analysis of iono-
phore polyethers [7]. This hypothesis suggests that an
achiral polyene undergoes sequential enantioselective
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epoxidation installing all required stereochemistries and
then the resulting polyepoxide serves as a substrate of
epoxide opening cascades to afford elaborated polyethers.
Taking into account for the remarkable stereochemical
regularity in ladder polyethers and oxasqualenoids, similar
cascade reactions are proposed in their biosynthesis [8,9].
Successful biomimetic synthesis of polycyclic polyether
systems further supports this attractive model [10]. How-
ever, elucidation of the enzymatic polycyclic polyether
formation mechanism has been a long-standing controver-
sial issue in organic chemistry.
The first breakthrough for this unsettled issue on poly-
cyclic polyether skeletal construction came in 2001 with
identification of a monensin biosynthetic gene cluster
achieved by Leadlay and co-workers [11]. They found a
flavin containing monooxygenase (EPX) monCI and a pair
of epoxide hydrolase (EH) monBI and monBII as candidate
genes involved in key epoxidation and epoxide opening
reactions, respectively. Gene inactivation studies revealed
that a single MonCI and a pair of MonBI/MonBII partici-
pate in three rounds of epoxidations and epoxide opening
reactions to afford monensin skeleton [12,13].
Among various ionophore polyethers reported to date,
lasalocid A (1A) has one of the simplest polyether com-
posed of THF and THP rings [14] which are presumably
constructed by the intriguing regioselective epoxide
opening reactions of bisepoxyprelasalocid (3A). In
addition, unlike other ionophore polyethers such as mon-
ensin, the chemical structure of lasalocid indicates that
the expected epoxidation and cyclization of prelasalocid
(2A) directly occur just after polyketide backbone assem-
bly (Figure 2). For these reasons, we focused on lasalocid
A as a model polycyclic polyether to investigate catalytic
mechanism of EPX and EH. Within past five years,
enzymatic polyether construction has been addressed
by pioneering functional analysis of a single flavin
containing monooxygenase Lsd18 and an epoxide hydro-
lase Lsd19 involved in lasalocid biosynthesis
[15,16,17,18,19,20,21]. In this review, we describe
mechanistic investigation of ionophore polyether lasalo-
cid biosynthesis and briefly consider the unified model for
natural polycyclic polyether biosynthesis.
Regioselective epoxide opening cascades
After identification of lasalocid biosynthetic gene cluster
and a homolog of epoxide hydrolases MonBI/BII, Lsd19
[15,16], lacking precedent information on an enzymatic
intramolecular ring closure reaction of hydroxyepoxide
Current Opinion in Chemical Biology 2013, 17:555–561
556 Mechanisms

Figure 1

OH
OH
O HO O H O
OH O H OH O H
CO2H H CO2H
OH lasalocid A OH isolasalocid

HO
H O OH
O
HO O O
H O O H O H O H O H H OH O H
O H O
HO OH OH H
OMe salinomycin
monensin A
O
HO
O O O
HO H H H H OH
OH
H H
O teurilene
O O
H H HO CHO
OH HO
O
squamocin B H O O H
H H
HO
O O H
H H OH
O H
O O brevetoxin B
O O O H H H
H H H
Current Opinion in Chemical Biology

Chemical structures of representative polycyclic polyether natural products.

Figure 2

R 6 Ph
10 12

1 O N H
OH O
4 CO2H O
O O
OH
A B C D

(R,R)-epoxide (R,R)-epoxide

24 Lsd18 O Lsd18 O O
R 15 R R
18 22
19 23
OH 2 OH 3 OH 4

OH Lsd19B Lsd19A
R R O
H O H O H O
H OH 5
1 O
non-enzymatic
reaction OH OH
R OH
or
H O H O
acid treatment 7 8

6
Current Opinion in Chemical Biology

Enzymatic catalysis for lasalocid polyether construction with Lsd18 and Lsd19.

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 Biosynthetic machinery of ionophore polyether lasalocid Minami et al. 557

led us to examine detection of enzymatic activity by
searching suitable substrates for Lsd19. Although we
expected that a single enzyme Lsd19 catalyzes both
THF and THP formations and thus it shows reasonable
substrate tolerance as in the case of limonene 1,2-epoxide
hydrolase and murine soluble EH [22,23], we chose a
putative intermediate bisepoxyprelasalocid (4A) pro-
posed by Westley [14] as a substrate to secure detection
of the cyclization activity.
Bisepoxyprelasalocid 4A was faithfully synthesized in a
stereocontrolled manner [24]. In the reaction with the
synthetic 4A, the recombinant Lsd19 catalyzes two
rounds of epoxide opening reactions in a highly regiose-
lective manner to afford lasalocid (1A) via monoether
intermediate 5A [17]. In contrast, incubation of 4A
without Lsd19 gave only isolasalocid (6A) by non-enzy-
matic 5-exo cyclizations. As expected, Lsd19 also cata-
lyzed rather simple substrates 4B, 4C and 4D mimicking
C15–C24 moiety of 4A to give THF–THP products 1 but
no conversion was observed in simple monoepoxide 7
[18]. Successful conversion of 5D suggested that mini-
mum structure for Lsd19 recognition is the terminal
hydroxyepoxide moiety with appropriate substitution at
C15. Interestingly, Lsd19 also accepts diastereomeric
bisepoxides [4C-a(unnatural) and 4C-b(natural)] and
converted them to the corresponding THF–THF and
THF–THP products, respectively with low efficiency
(Figure 3a) [18]. Brief discussion of the remarkable
regioselectivities is described below.
The regioselective intramolecular ring closure reaction of
hydroxyepoxide is one of the intriguing topics in the
polycyclic polyether construction. In Lsd19 catalyzing
reaction, combination of energetically favored 5-exo cycli-
zation and energetically disfavored 6-endo cyclization [25]
gives THF–THP type polycyclic ether skeleton. There-
fore, Lsd19 is regarded as a model enzyme to shed light
on the regioselective cyclization and structural analysis of
Lsd19 was then carried out. The Lsd19 structure in
complex with substrate and product analogs has defined
the presence of pseudo-dimeric domain architecture com-
posed of structurally resembled N-terminal domain
(Lsd19A) and C-terminal domain (Lsd19B) [20]. Bisep-
oxide 4C and its cyclized product 6C are seen in the
active site of Lsd19A and Lsd19B, respectively

Figure 3

(a) Ph β Ph α
O O O O
O N O N

O O OH O O OH
4C-β: β-epoxide 4C-α: α-epoxide

(b) Lsd19A Lsd19B (c)

catalytic pair O
Lsd19A: D38-E65
O D or E
D38 Lsd19B: D170-E197
D170 H
O
R1 R2
5-exo
O O
E65 Y251 H 6-endo
D or E O
E197
(d) R
R
N N O H
O2 N N O
NH
N NH
H O N
O O step 2 H NAD+
O
H
FAD-4a-OOH FADH2 step 1 Lsd18
step 3 Lsd18
R
N N O
NADH
NH
N
O O
FAD
Current Opinion in Chemical Biology

(a) Substrate analogs for Lsd19 catalyzed epoxide opening reaction, (b) closed view of Lsd19A and Lsd19B active sites, (c) catalytic mechanism in
Lsd19 cyclization, and (d) catalytic mechanism in Lsd18 epoxidation.

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558 Mechanisms
(Figure 3b). The observed structure suggested that each
domain independently participates in a single cyclization
reaction. This hypothesis was confirmed by domain dis-
section analysis [19].
The closed view of Lsd19A active site indicated that the
hydroxyl group of 4C interacts with Asp38, whereas
epoxide lies within hydrogen bonding distance to
Tyr14 and Glu65. The similar view was observed in
the active site of Lsd19B in complex with 6C, and
Asp170 and Glu197 were regarded as catalytic residues.
Importance of these acidic amino acid pair for the catalyst
was demonstrated by extensive mutational analysis [19].
The corresponding acidic amino acid pair found in Lsd19
is highly conserved in other EHs involved in the biosyn-
thesis of monensin (mon) [11], nanchangmycin (nan) [26],
nigericin (nig) [27], and salinomycin (sal) [28,29]. Thus, all
EHs share the common catalytic mechanism; Asp is acted
as a base which abstracts the hydrogen atom from the
hydroxyl group of the substrate and the other Glu act as a
general acid to activate the epoxide moiety by protona-
tion (Figure 3c). The similar hydrolysis mechanism invol-
ving acid/base catalysis was proposed in the reaction by
limonene 1,2-epoxide hydrolase [30].
Identification and structural analysis of Lsd19B set the
stage to identify an important factor to control the regios-
electivity in epoxide opening reaction. ‘Theozyme’ cal-
culations, optimizing the competing 5-exo and 6-endo
transition structures surrounded by fixed catalytic resi-
dues (Asp170 and Tyr251), revealed preference of 6-endo
cyclization in 2.5 kcal mol 1 [20]. This is sufficient to
account for exclusive formation of lasalocid in preference
to isolasalocid. Therefore, active site pre-organization
Figure 4
(12R, 13R) (20S, 21S)
HO (16R, 17R)
O O O O
13 17 20
9 9
12 16 21
H OH
OH
O OH
S 13
Enz
H MonBII
monensin
O 20
H HO
Cooperative catalysis in monensin EH epoxide opening reaction.
Current Opinion in Chemical Biology 2013, 17:555–561
may be a key factor in enzymatic control of the second
6-endo cyclization. This is presumably supported by the
fact that regioselectivity change (5-exo to 6-endo cycliza-
tion) occurred in the reaction with unnatural diastereo-
meric epoxide 4C-a due to preventing correct binding of
the hydroxyepoxide moiety. Further mechanistic
analyses based on the structural, mutational, theoretical
studies may provide an opportunity for artificial manip-
ulation of Lsd19 function to produce ladder-type poly-
ether natural product.
The first biochemical and structural evidence described
above indicated that each EH domain independently
catalyzes a single cyclization in lasalocid biosynthesis.
This single EH/single cyclization protocol cannot be
applied to the biosynthesis of ionophore polyethers such
as monensin and nigericin because the number of EH
domain/subunits (monensin; MonBI/MonBII, nigericin;
NigBI/NigBII) is less than that of the epoxide opening
reactions (monensin, nigericin; three rounds of cyclization
required) (Figure 4) [7]. Database analysis revealed that
MonBI and MonBII have moderate sequence similarity
with Lsd19B (45% identity) and Lsd19A (42% identity),
respectively. Alignment of these EHs showed acidic
residues predicted catalysis, and homology-modeling
study indicated that MonBI (330 Å) and MonBII
(405 Å) have large active site compared with Lsd19B
(240 Å) and Lsd19A (390 Å), reflecting difference of
the substrate chain length. On the basis of the bioinfor-
matics analysis, it is speculated that MonBI catalyzes the
terminal cyclization and MonBII does the internal two
rounds of cyclizations [20]. Recent biochemical study
with substrates mimicking the internal olefin showed that
the enzymatic conversion of substrate analogs required
HO
OH O
O H O
MonBI
HO
O MonBII O
9
1st step O 12 OH
H O
MonBI MonBI
OH O MonBII
21 17
16
3rd step O 2nd step
H
O
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 Biosynthetic machinery of ionophore polyether lasalocid Minami et al. 559

both EHs, suggesting unexpected cooperative effect of
MonBI and MonBII [31]. Currently, we speculated that
MonBI and MonBII form heterodimeric structure as
Lsd19 does, and that in this structure, one of the EHs
may function as an allosteric regulator for multiple cat-
alysis of the other EH. Further experimental results
would provide insight into the complicated regulation
system found in monensin epoxide opening reactions.
Sequential and stereoselective epoxidation
Unlike EH Lsd19, Lsd18 is a flavin containing mono-
oxygenase and thus Lsd18 catalyzing epoxidation
requires several co-factors (such as NAD(P)H, FAD,
FMN) and co-factor recycling system (flavin reductase)
[32]. A major drawback of the reaction by this type of
monooxygenase is requirement of labile 4a-hydroperoxy-
flavin as an actual oxidation species. This made difficult
to optimize the reaction conditions and to identify true
substrate at the same time. To avoid these difficulties, an
alternative strategy applying transformation with micro-
organism introduced the corresponding gene was
examined in the functional analysis of Lsd18. Rhodococcus
species which exhibits resistance toward organic solvents
and an efficient conversion in many cases [33,34] was
selected as the first candidate to be examined [21].
When simple olefin 8 mimicking C19–C24 moiety of
possible precursor 2A was incubated with Rhodococcus
transformant harboring lsd18 gene, extremely rapid con-
version of 8 to the corresponding (R,R)-epoxide in a
highly stereoselective manner was observed (40% sub-
strate consumption within 20 min). Thus, this simple
system was effective to detect the epoxidation activity
and to facilitate the screening of substrate analogs.
epoxidation reaction catalyzed by not only Lsd18 but
also other EPXs, because two FAD binding motifs,
GxGxxG and GD [38,39], are highly conserved in all
EPXs for ionophore polyether biosynthesis.
As exemplified in the Lsd18 catalyzing epoxidation,
highly stereoselective epoxidations of the internal tri-
substituted olefins with the monensin epoxidase MonCI
also occur in the biosynthesis of monensin-type iono-
phore polyether affording (R,R)-epoxides while the
MonCI of the terminal disubstituted olefin gives (S,S)-
epoxide (Figure 4). The stereochemical reversal in the
MonCI epoxidation is remarkable but origin of selectivity
is currently unknown. Answer for the structural require-
ment for substrate recognition will be given when the X-
ray crystal structural analysis of MonCI complex with
simple or truncated analogs is available.
Biosynthetic proposal
Recently, artificial cleaving method with malonyl-CoA
analog was applied to study the lasalocid biosynthesis
[40]. Extensive analysis of cleavage products allowed
detecting several intermediate analogs with polyether
skeleton, suggesting that the construction occurs during
Figure 5
PKS S
O O OH O HO
Lsd18

Biotransformation system was then applied to the

O
sequential epoxidation of diene 2B which has an identical PKS S
partial structure of putative intermediate 2A [21]. Time O O OH O HO
course analysis showed rapid consumption of 2B and
spontaneous formation of monoepoxide 3B and hydro- Lsd18
xyethers (5B, 6B) in a highly stereoselective manner,
indicating that initial epoxidation occurred at the terminal PKS S
O O
olefin (Figure 2). The enzymatic epoxidation of 2B was
also demonstrated by in vitro reaction with the recombi- O O OH O HO
nant Lsd18, which was purified as a FAD bound form. Lsd19A
This established cofactor preference to NADH and dis-
tinct regeneration activity of reduced flavin without a
flavin reductase, which is a universal enzyme catalyzing PKS S O
O
FAD reduction by NAD(P)H (Figure 3e). On the basis of O O OH O H
OH
structural insights of functionally and sequentially related
Lsd19B
monooxygenases such as p-hydroxybenzoate hydroxylase
( pHBH), TetX and RebC [35,36,37], it was confirmed
that is oalloxazine ring of FAD moves from the OUT PKS S OH
position to IN position during their catalysis for the O HO
O O OH O H
oxidation/reduction sequence of FAD. The mobile H
FAD is thought to be a key factor to complete the Current Opinion in Chemical Biology

catalysis of redox process in the single active site. Similar

‘mobile flavin’ mechanism may be applied to the Detailed reaction sequence in the late stage biosynthesis of lasalocid.

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560 Mechanisms
polyketide chain extension. Taking into consideration of
these results, we propose a detailed biosynthetic mech-
anism of lasalocid (Figure 5). A sequential Lsd18 epox-
idation of PKS bound diene intermediate proceeds from
terminal to internal olefin followed by Lsd19 catalyzing
regioselective cyclization of the resultant hydroxyepoxide
in the reverse direction. Microorganisms choose this
reaction sequence presumably for preventing the non-
enzymatic 5-exo cyclizations. We believe this strategy is
versatile for all ionophore polyether biosynthesis because
the corresponding EH domains and EPX are found in
their gene clusters. Interestingly, an alternative mechan-
ism of mutiple catalysis of EPX and EH involving two
rounds of stepwise epoxidation/cyclization has been
reported in the meroterpenoid paxilline [41].
Concluding remarks
Compared with chemical synthesis, nature chooses very
elegant and sophisticated strategy to synthesize highly
elaborated polycyclic polyether skeleton with a small
number of EPX and a pair of EH. The unique and
fascinating function of these enzymes would attract
researchers in not only bioorganic but also synthetic
chemistry fields. Recent five years studies on ionophore
polyethers allow us to draw detailed and rational mech-
anism. However, unanswered questions remains to be
elucidated: firstly, enantiofacial reversal of premonensin
epoxidation [(R,R)-epoxides for 12,13-olefin and 16,17-
olefin; (S,S)-epoxide for 20,21-olefin]; secondly, allosteric
regulation in monensin EH-catalyzed cyclizations;
thirdly, EH reaction accompanied with spiroketal for-
mation (monensin and salinomycin). These can be
addressed by applying the biotranformation system for
EPX and in vitro analysis utilizing simple substrate har-
boring key functionalities. Further detailed study on
characterization of enzymatic catalytic mechanism for
polycyclic polyether construction may be important to
engineer these enzymes and to provide the artificial
polycyclic polyethers.
On the basis of its remarkable substrate tolerance and
enantioselectivity in disubstituted and trisubstituted ole-
fins, EPX in polyether biosynthesis is an attractive cat-
alyst for asymmetric epoxidation. Thus, structural study
on the factor which determines enantiofacial selectivity in
Lsd18 is important for its application. In addition, concise
synthesis of medium size cyclic-ether from the corre-
sponding polyolefin is still challenging task. Thus, enzy-
matic transformation of artificial substrates with EPX and
EH would offer chemo-enzymatic synthesis of target
natural polyether derivatives in an efficient manner.
Acknowledgements
The authors gratefully acknowledge all collaborators mentioned in
references. This work was supported by MEXT Research Grant on
Innovative Area 22108002 to H. Oikawa.
Current Opinion in Chemical Biology 2013, 17:555–561
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Current Opinion in Chemical Biology 2013, 17:555–561