Sequential enzymatic epoxidation involved in polyether lasalocid biosynthesis

Article abstract of DOI:10.1021/ja301386g

Enantioselective epoxidation followed by regioselective epoxide opening reaction are the key processes in construction of the polyether skeleton. Recent genetic analysis of ionophore polyether biosynthetic gene clusters suggested that flavin-containing monooxygenases (FMOs) could be involved in the oxidation steps. In vivo and in vitro analyses of Lsd18, an FMO involved in the biosynthesis of polyether lasalocid, using simple olefin or truncated diene of a putative substrate as substrate mimics demonstrated that enantioselective epoxidation affords natural type mono- or bis-epoxide in a stepwise manner. These findings allow us to figure out enzymatic polyether construction in lasalocid biosynthesis.

Full text of DOI:10.1021/ja301386g

Communication
pubs.acs.org/JACS
Sequential Enzymatic Epoxidation Involved in Polyether Lasalocid
Biosynthesis
Atsushi Minami,† Mayu Shimaya,† Gaku Suzuki,† Akira Migita,† Sandip S. Shinde,† Kyohei Sato,†
Kenji Watanabe,‡ Tomohiro Tamura,§ Hiroki Oguri,† and Hideaki Oikawa*,†
†Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan
‡Division of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan
§Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo 062-8517,
Japan
S
* Supporting Information
ABSTRACT: Enantioselective epoxidation followed by
regioselective epoxide opening reaction are the key processes
in construction of the polyether skeleton. Recent genetic
analysis of ionophore polyether biosynthetic gene clusters
suggested that flavin-containing monooxygenases (FMOs)
could be involved in the oxidation steps. In vivo and in vitro
analyses of Lsd18, an FMO involved in the biosynthesis of
polyether lasalocid, using simple olefin or truncated diene of a
putative substrate as substrate mimics demonstrated that
enantioselective epoxidation affords natural type mono- or
bis-epoxide in a stepwise manner. These findings allow us to
figure out enzymatic polyether construction in lasalocid bio-
synthesis.
Figure 1. (A) Enzymatic polyether formation catalyzed by Lsd18 and
Lsd19 in lasalocid biosynthesis. (B) Compounds tested as substrate
mimic for Lsd18 reaction.
onophore polyethers, a structurally unique group of natural
polyketides, has a polycyclic ether skeleton with multiple
I
stereocenters.1 Structural diversity of these natural products is
derived from combination of the number and size of ether
rings. In 1983, Cane, Celmer, and Westley proposed a unified
biosynthetic model (CCW model) for polyether construction
in which epoxidation of the linear polyene intermediate in a
stereoselective manner followed by regioselective cascade
cyclization provided these polyethers.2
to the second step of polyether formation, initial sequential
epoxidation, which is an important step for introducing multi-
ple chiral centers, has not been investigated in vitro and its
detailed mechanism has not yet been determined. In this paper,
we describe the enzymatic epoxidation with Lsd18, a flavin-
containing monooxygenase (FMO), involved in lasalocid biosyn-
thesis (Figure 1A).
FMO catalyzes various reactions, including epoxidation in which
FMOs utilize 4a-hydroperoxyflavin as an electrophile.12 As this
oxidant is prepared from reduced flavin and molecular oxygen,
reduction of an oxidized flavin is one of the key processes in FMO-
catalyzed reactions. For this purpose, flavin reductases, such as
SsuE13 and Fre,14 are frequently used as alternatives to the native
reductase. To detect enzymatic activeity of Lsd18 in a simple
system, we conducted biotransformation studies using a Rhodo-
coccus host, which exhibits resistance toward organic solvents and is
therefore an industrially important genus of actinomycetes.15
The lsd18 gene was cloned into pTipQC216 and expressed in
Rhodococcus erythropolis L-88. As expected, Lsd18 expression
was observed upon addition of thiostrepton (Figure S1). On
the basis of the results of a previous biotransformation study
After the pioneering work by Leadlay and co-workers on
identification of monensin biosynthetic gene cluster,3 a series of
gene inactivation experiments established the involvement of
polyene and polyepoxide intermediates in polyether construc-
tion.4,5 Thus, the CCW model consisting of epoxidation and
epoxide-opening reaction was firmly established. Recently, the
second step of polyether formation has been studied extensively
by our group. We found that recombinant epoxide hydrolase
Lsd19 catalyzes energetically disfavored conversion of the chemi-
cally synthesized putative precursor, bisepoxide of prelasalocid (2), to
lasalocid (1) in a highly regioselective manner and that this epoxide
hydrolase shows promiscuous substrate specificity (Figure 1A).6−8
More recently, we proposed a detailed reaction mechanism based on
biochemical analysis and solved the structure of Lsd19 complexed
with a substrate analogue.9,10 This allowed us to propose the
mechanism of anti-Baldwin cyclization catalyzed by Lsd19. Applying
this established procedure, we also characterized the function of
epoxide hydrolases involved in monensin biosynthesis.11 In contrast
Received: February 10, 2012
Published: April 17, 2012
© 2012 American Chemical Society
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Communication
Figure 2. HPLC profiles of the MNPA derivatives. (A) Derivatives from Lsd18 catalyzed reaction product 5, (B) mixture of four possible derivatives
(7a−7d). To simplify the discussion on Lsd18 catalyzed epoxidations, we used the same numbering system of substrate analogues as that of
prelasalocid 2.
with polyether-producing organisms and Streptomyces host with
a putative epoxidase gene,17 we initially tested ( )-linalool 3 as
a substrate in our biotransformation system (Figure 1B). GC−MS
analysis of the extracts from whole-cell reaction mixture
revealed 65% conversion to linalool oxide (syn/anti = 1/1)
after 49 h of incubation (Figure S2). Formation of linalool
oxide only in the induced cells indicated that trisubstituted
olefin was epoxidized and spontaneous cyclization occurred,
strongly suggesting that Lsd18 epoxidized 3. Efficient
conversion in this system allowed simple screening of substrate
analogues (Figure S3). Therefore, we employed the reaction
with a simple substrate 4 possessing terminal trisubstituted
olefin moiety mimicking the C19−C24 part of 2 (Figure 1B).
LC−MS analysis revealed that 4 was epoxidized to afford 5 and
6 after 60 min of incubation. To determine the stereoselectivity
in this epoxidation, reaction products were esterified with (R)-
2-methoxy-2-(1-naphthyl) propionate (MNPA) and the
resultant esters 7 were analyzed by HPLC.18 The results
showed two major peaks corresponding to authentic diastereo-
mers of 7a and 7b and one trace peak corresponding to 7d,
indicating that Lsd18-catalyzed epoxidation proceeded in a
highly stereocontrolled manner {6a, 99% ee; 6b, 87% ee}.
Determined absolute stereochemistry of 5a and 5b are identical
to those of the bisepoxide of 2 (Figure 2). Formation of 7d, an
unnatural (22S, 23S)-epoxide, suggests that C19 stereo-
chemistry of 4 affected stereoselectivity of Lsd18-catalyzed
epoxidation.
We next examined bioconversion of C12−C24 diene 8,
which has a structure identical to that of the C12−C24
moiety of 2 (Figure 1B).7,19 Time course analysis showed rapid
Figure 3. (A) Biotransformation experiment with 8 in Lsd18
consumption of 8 and spontaneous production of hydroxy-
ethers (11, 12) via mono- and bis-epoxide (Figure S4). After 6 h
of incubation, 8 was converted to monoepoxide 9 (10%),
monocyclic ether 11 (43%), and isolasalocid ketone 12 (47%)
(Figure 3A). Identification of 12 as a single diastereomer
indicated that Lsd18 catalyzed epoxidation in a highly enantio-
selective manner. In addition, acid treatment of the reaction
mixture predominantly gave diol S3 instead of tetrahydrofuran
S5 (Figure S5), suggesting that initial epoxidation occurred at
the terminal olefin.
transformant, (i) mass chromatogram (m/z 361), (ii) mass chromato-
gram (m/z 377) of the reaction products, (iii) mass chromatogram (m/z
377) after acid treatment, (iv) authentic 12. (B) In vitro experiment with
8 in co-incubation of Lsd18 and Lsd19, (i) mass chromatogram (m/z
345), (ii) mass chromatogram (m/z 361), (iii) mass chromatogram (m/z
377) of the reaction products, (iv) authentic 12 and 13.
examined. Lsd18 was expressed as an N-terminal His6-tagged pro-
tein, and purified by Ni-NTA column chromatography (Figure S6).
Solutions of the purified Lsd18 were yellow color and showed
characteristic UV−visible spectrum of flavoprotein at 362 and
451 nm (Figure S7). Denaturation of Lsd18 with methanol followed
To determine the cofactor requirements and detailed reaction
mechanism, enzymatic reaction with recombinant Lsd18 was
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Communication
by reverse-phase HPLC analysis showed a major peak identical to
FAD, indicating that FAD exists as a tightly bound form and
the molar ratio of Lsd18 and FAD was estimated as 1/0.82
(Figure S8).
terminal and internal olefin but the other two FMOs epoxidize
terminal olefin only in polyene precursor. The differences between
these epoxidases with regard to function are intriguing (Figure S15).
Recently, artificial cleavage with the malonyl CoA analogue
from the linear polyketide intermediates having different chain
lengths appended to lasalocid polyketide synthase (PKS) has
been reported.27 Extensive analysis of the cleavage products
indicated that polyene precursors bound to PKS are epoxidized
and cyclized to form a polyether skeleton. Preparation of these
intermediate appended to PKS and its in vitro experiment
would be difficult. However, based on our experimental results
regarding Lsd18 and Lsd19, in vitro analysis using model sub-
strates would be useful for investigating the intriguing epoxidation
and epoxide-opening cascade. In addition, this finding also
suggests that only a small number of epoxidases and epoxide
hydrolases are required to install multiple chiral centers in enzy-
matic synthesis of the polyether skeleton. As installation of
multiple chiral centers requires multistep transformations in total
synthesis of natural polyethers, including monensin28 and
brevetoxin,29 enzymatic construction of the polyether skeleton is
very attractive. To verify this hypothesis, we are currently working
on analysis of a much more complex system.
Then, epoxidation of 8 with Lsd18 was carried out in the
presence of NAD(P)H, FAD, and flavin reductase under various
conditions (Figures S9, S10). In the time course experiments,
Lsd18 reaction with NADH afforded monoepoxide 9 in higher
conversion rate than that of NADPH (Figure S10 (D) vs (E),
Figure S11). The preference of Lsd18 to NADH was also
supported by rapid decrease of NADH compared with NADPH in
monitoring of absorbance at 340 nm (Figure S12 (A) vs (B)).
Exogenous FAD was not necessary when using freshly prepared
Lsd18, but addition of FAD was effective in maintaining the
epoxidation activity (Figure S10 (F) vs (G) vs (A)). These results
indicate that epoxidase Lsd18 can reduce FAD using NADH.
However, addition of flavin reductase Fre to the reaction mixture
increased production of 9 (1.5 times increase) (Figure S10 (A) vs
(H)). In the presence of Fre, complete consumption of NADH
(within 6 min) was observed (Figure S12 (C)), indicating the
formation of reduced FAD although it is not completely correlated
with epoxidation. The results shown above suggest that a tem-
porary increase of the reduced FAD in the reaction mixture with
Fre caused its saturation in the active site of Lsd18 and this allows
effective turnover compared with the reaction without Fre. How-
ever, the effect of Fre for the epoxidation reaction was limited
because rapid consumption of NADH simply increased
concomitant decomposition of direct reactive species, 4a-hydro-
peroxyflavin, derived from the reduced FAD. Applying the establis-
hed conditions in sequential reaction of Lsd18 and Lsd19 with 8,
two products were detected on LC−MS analysisthe expected
product 9 (52%) and lasalocid ketone 13 (13%), which was
identical to the authentic sample (Figure 3B, Figure S13).
Motif analysis of Lsd18 with Pfam20 revealed that it has the
FAD binding-3 motif similar to other FMOs, such as p-hydro-
xybenzoate hydroxylase and TetX.21,22 The biochemical pro-
perties of Lsd18 also corresponded to these enzymes. There-
fore, Lsd18 could be classified into subclass A catalyzing two
half reactions in the single polypeptide:12 (1) reductive half-
reaction using NAD(P)H and oxidized FAD to give reduced
FAD and (2) oxidative half-reaction using the reduced FAD
and molecular oxygen to afford 4a-hydroperoxyflavin followed
by epoxidation of the substrate (Figure 4). A similar epoxidation
In conclusion, we established an in vivo rapid screening system
for substrates of epoxidation using Rhodococcus host. This enabled
us to find epoxidase activity for Lsd18 against a simple olefin or
truncated diene substrates as substrate mimic. Sequential epoxida-
tions of the diene and predominant formation of a product with
the correct absolute configurations strongly support the role of
Lsd18 in lasalocid biosynthesis.
ASSOCIATED CONTENT
* Supporting Information
Additional information as noted in text. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
hoik@sci.hokudai.ac.jp
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Grants-in-Aid for Scientific
Research [22108002 to H. Oikawa] from Japan Society for the
Promotion of Science (JSPS).
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