Biosynthesis of acaterin: Mechanism of the reaction catalyzed by dehydroacaterin reductase

Article abstract of DOI:10.1016/j.bmc.2006.05.039

Dehydroacaterin reductase is an enzyme which catalyzes the final step of acaterin biosynthesis, that is, the reduction of the C-4/C-5 double bond of dehydroacaterin. The mechanism of the reduction was investigated with a cell-free preparation obtained from the acaterin-producing microorganism, Pseudomonas sp. A 92. Incubation of dehydroacaterin in the presence of [4,4-²H₂]NADPH or D₂O followed by ²H NMR analysis of the resulting acaterin revealed that the deuterium atom from NADPH was incorporated into the C-5 position of acaterin, while the deuterium atom from D₂O was introduced into the C-4 position. It was further demonstrated that the pro-R hydrogen at C-4 of NADPH was stereospecifically utilized in this reduction.

Full text of DOI:10.1016/j.bmc.2006.05.039

Bioorganic & Medicinal Chemistry 14 (2006) 6404–6408
Biosynthesis of acaterin: Mechanism of the reaction catalyzed
by dehydroacaterin reductase
Sayaka Nakano, Wataru Sakane, Hiroshi Oinaka and Yoshinori Fujimoto*
Department of Chemistry and Materials Science, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo 152-8551, Japan
Received 1 May 2006; revised 19 May 2006; accepted 20 May 2006
Available online 9 June 2006
Abstract—Dehydroacaterin reductase is an enzyme which catalyzes the final step of acaterin biosynthesis, that is, the reduction of
the C-4/C-5 double bond of dehydroacaterin. The mechanism of the reduction was investigated with a cell-free preparation obtained
from the acaterin-producing microorganism, Pseudomonas sp. A 92. Incubation of dehydroacaterin in the presence of
[4,4-2H2]NADPH or D2O followed by 2H NMR analysis of the resulting acaterin revealed that the deuterium atom from NADPH
was incorporated into the C-5 position of acaterin, while the deuterium atom from D2O was introduced into the C-4 position. It was
further demonstrated that the pro-R hydrogen at C-4 of NADPH was stereospecifically utilized in this reduction.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
utilizes a glyceric acid equivalent as the immediate bio-
synthetic precursor of the branched C3 unit of 1.7 These
accumulated evidence led us to postulate biosynthetic
route for acaterin (Scheme 1). More recently we have
developed a cell-free system catalyzing transformation
of 2 to 1 (the enzyme was named dehydroacaterin reduc-
tase) from the acaterin-producing microorganism. The
availability of the in vitro system prompted us to inves-
tigate the mechanism of the reduction of the unique sub-
strate, an a, b:c, d-diene system conjugated with a
lactone carbonyl group. This paper describes the regio-
specificity of hydrogen transfer from NADPH and
water, and the stereospecificity in the utilization of the
prochiral C-4 hydrogens of NADPH in the reaction cat-
alyzed by dehydroacaterin reductase.
Acaterin (1) was isolated from Pseudomonas sp. A 92 as
an inhibitor of acyl-CoA:cholesterol acyltransferase.1
Dehydroacaterin (2) was subsequently isolated by our
group and elucidated to be the immediate biosynthetic
precursor of acaterin.2 We have been studying the bio-
synthesis of acaterin, since little has been known on
the biosynthesis of secondary metabolites with a 2-pen-
ten-4-olide skeleton.3 In the previous papers, we demon-
strated that glycerol was incorporated into the branched
C3 moiety (C-3, -4, and -5 positions) in such a manner
that sn-C-1 of glycerol becomes C-3 of 1,4 and pro-R
and pro-S hydrogens at sn-C-3 of glycerol become 5E
and 5Z hydrogens, respectively, of 4-dehydroacaterin
(2).5 Furthermore, hydrogen atoms at sn-C-1 of glycerol
were lost completely during the transformation.4 On the
basis of these findings, we proposed that the immediate
precursor of the branched C3 unit of 1 could be a glycer-
ic acid equivalent, and a tetronic acid-type intermediate
would be involved in the formation of 3-H type com-
pounds.4 Moreover, we reported evidence supporting a
coupling of octanoate and a C5 unit corresponding to
the lactone part in acaterin biosynthesis.6 In addition,
we indicated that the biosynthesis of agglomerins,
which belong to the same class of secondary metabolites,
2. Results and discussion
The active cell-free preparation (20,000g supernatant)
was obtained by sonicating the cells harvested in a phos-
phate buffer (pH 7.4) followed by centrifugation of the
sonicate. Addition of NADPH showed increased activi-
ty for the conversion of 2 to 1. The enzyme activity was
conveniently determined by reversed-phase HPLC anal-
ysis of the product formed.
Dehydroacaterin was incubated with the cell-free prepa-
ration in the medium of D2O/H2O (2:1) in the presence
of NADPH. The resulting acaterin was purified and
analyzed by 2H NMR spectroscopy. The 2H NMR
Keywords: Acaterin; Dehydroacaterin; Dehydroacaterin reductase;
Michael addition; Conjugated carbonyl.
*
Corresponding author. Tel./fax: +81
fujimoto@cms.titech.ac.jp
3
5734 2241; e-mail:
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.05.039

S. Nakano et al. / Bioorg. Med. Chem. 14 (2006) 6404–6408
COSCoA
6405
HOOC
O
O
O
n-C7H15COSCoA
n-C7H15
O
O
H
O
PO C
OH
OP
– POH
HO
HO
OP
OH
OH
O
O
O
O
O
1'
n-C7H15
1
dehydroacaterin
reductase
2
n-C7H15
n-C7H15
O
O
3
4
5
1
2
Scheme 1. Postulated biosynthesis of acaterin (1). The present studies deal with dehydroacaterin reductase which catalyzes the conversion of
dehydroacaterin (2) to 1.
spectrum is shown in Figure 1. The deuterium signal res-
onated at d 5.07 corresponds to the signal of H-4 of 1,
thus indicating that the deuterium atom was introduced
at C-4 of 1 regiospecifically. Dehydroacaterin was then
incubated in the presence of [4,4-2H2]NADPH with the
cell-free preparation. The formed acaterin showed a
hydrogen at C-4 of NADPH is utilized in this reduction.
Incubation of dehydroacaterin in the presence of [4-pro-
R-2H]- or [4-pro-S-2H]NADPH cofactors would solve
the question. Generally, the requisite [4-pro-R-2H]
NADPH has been prepared enzymatically from
NADP+ with an [2H]alcohol as a deuterium source in a
reaction catalyzed by an alcohol dehydrogenase such as
Thermoanaerobium brockii alcohol dehydrogenase,8
while [4-pro-S-2H]NADPH has been obtained with
[1-2H]glucose as a deuterium source in a reaction cata-
lyzed by glucose dehydrogenase such as Bacillus megate-
rium glucose dehydrogenase.9 Mosted et al. reported an
unprecedented A-specific glucose dehydrogenase, Ther-
moplasma acidophilium glucose dehydrogenase,10 and
now this enzyme is commercially available. In the present
study, [4-pro-R-2H]- and [4-pro-S-2H]NADPH cofactors
were enzymatically prepared from NADP+ with
2
signal at d 1.45 in the H NMR spectrum, which corre-
sponds to 5-H3 of 1, thus indicating that the deuterium
atom from NADPH was incorporated regiospecifically
into the C-5 position of 1. These results agree with the
Michael addition mechanism wherein the hydride trans-
fer from NADPH took place at the C-5 position and the
resulting enolate anion was trapped by the proton from
water at the C-4 position.
With the hydride transfer from NADPH having been
established, we further investigated which prochiral
Figure 1. 2H NMR spectra (61 MHz, CHCl3) of acaterin obtained from dehydroacaterin with a cell-free preparation. Upper: incubated in D2O/H2O
in the presence of NADPH. Middle: incubated in H2O in the presence of [4,4-2H2]NADPH. Lower: 1H NMR spectrum of non-labeled acaterin.

6406
S. Nakano et al. / Bioorg. Med. Chem. 14 (2006) 6404–6408
cofactors. The resulting acaterin was purified and ana-
lyzed by H NMR spectroscopy. As shown in Figure
2
3, acaterin obtained in the presence of [4-pro-
R-2H]NADPH showed a deuterium peak at d 1.45, while
2
instead no signal was detected near d 1.45 in the H
NMR spectrum of acaterin obtained in the presence of
[4-pro-S-2H]NADPH. It is therefore concluded that
dehydroacaterin reductase catalyzes stereospecific trans-
fer of the 4-pro-R hydrogen (A-specific) of NADPH to
the C-5 position of dehydroacaterin.
3. Conclusion
The present study established the regiochemistry of the
introduction of hydrogen atoms from NADPH and
water in the dehydroacaterin reductase-catalyzed reduc-
tion. In addition, it was determined that the pro-R
hydrogen at C-4 of NADPH (A-specific) was utilized
stereospecifically in this reduction. To our knowledge,
regiochemistry in the reduction of a,b;c,d-unsaturated
carbonyl compounds has been studied only in one case.
The reduction of (E,E)-2,4-dienyoyl CoA catalyzed by
2,4-dienoyl CoA reductases from Escherichia coli and
bovine liver mitochondria was reported to proceed
according to the Michael addition mechanism wherein
the hydride attacks the C-5 position of the substrate.11,12
Interestingly, it was reported that in the case of E. coli
(E,E)-2,4-dienoyl CoA reductase neither pro-R nor
pro-S hydrogen of NADPH was incorporated into the
product.12 The crystal structure of the E. coli reductase
(an iron–sulfur flavoprotein) was recently published13
and the reason the C-4 hydrogen of NADPH is not
directly transferred into the substrate was postulated.
In contrast, it was reported that the pro-S hydrogen at
Figure 2. 1H NMR spectra (500 MHz, D2O) of the C-4 hydrogen of
[4-pro-S-2H]- and [4-pro-R-2H]NADPHs.
[1-2H]glucose as a common deuterium source by the
action of T. acidophilium10 (A-specific) and Cryptococus
uniguttulas10 (B-specific) glucose dehydrogenases, respec-
1
tively. The H NMR spectra of the labeled NADPH
molecules are shown in Figure 2, which indicated excel-
lent stereochemical purity at the C-4 position.10
Meanwhile, purification of dehydroacaterin reductase
has progressed to some extent. Thus, the cell-free prep-
aration was fractionated by (NH4)2SO4 precipitation
and the precipitates were further fractionated through
a DEAE column to give a partially (16-fold) purified
dehydroacaterin reductase fraction. Dehydroacaterin
was then incubated with this enzyme preparation in
the presence of [4-pro-R-2H]- or [4-pro-S-2H]NADPH
Figure 3. 2H NMR spectra (76 MHz, CHCl3) of acaterin obtained from dehydroacaterin upon incubation with a partially purified enzyme solution.
Upper: incubated in the presence of [4-pro-R-2H]NADPH. Middle: incubated in the presence of [4-pro-S-2H]NADPH. Lower: 1H NMR spectrum of
non-labeled acaterin.

S. Nakano et al. / Bioorg. Med. Chem. 14 (2006) 6404–6408
OH
6407
HR
HS
O
O
CONH2
N
N
n-C7H15
O
NH
N
N
HR
H
O
(from H2O)
NADPH
HS
Enz-FAD
H
1
OH
O
O
CONH2
N
N
n-C7H15
O
+
N
NH
N
HR
O
NADP+
2
Enz-FADH2
Scheme 2. Reactions catalyzed by dehydroacaterin reductase.
C-4 of NADPH was stereospecifically transferred direct-
ly to C-5 of the substrate in the reaction catalyzed by the
bovine enzyme, which is free from FAD.11 The deute-
ride transfer from [4-pro-R-2H]NADPH to acaterin pro-
ceeded practically in a stoichiometric manner, as
cultures of the strain, grown in nine 500-mL baffled
Erlenmeyer flasks, each containing 100 mL of liquid
medium, were harvested by centrifugation (4000g,
10 min at 4 ꢁC). The cells (wet wt., 6.4 g) were washed
with 0.1 M sodium phosphate buffer (pH 7.4), resus-
pended in the phosphate buffer (20 mL), and sonicated
with a Bronson Sonifier 250 for 10 min · 3 times at
0 ꢁC (ice water bath). The sonicate was volumed up to
60 mL and centrifuged at 8000g for 10 min. The super-
natant was then centrifuged at 20,000g for 30 min and
the resulting supernatant was used as the cell-free prep-
aration (60 mL, 22 mg/mL protein). A portion of this
preparation was used for the study to elucidate the reg-
iospecificity of the reaction. Enzyme activity was deter-
mined by incubating dehydroacaterin in an enzyme
solution followed by analyzing an aliquot of the AcOEt
extract by HPLC (Shimadzu Shim-Pack CLC ODS col-
umn 15 cm · 0.6 cm i.d.; solvent, MeOH/H2O = 6:1;
flow rate 1.0 mL/min; UV detection at 230 nm; typical
retention times for 1 and 2 were 4.5 and 5.1 min,
respectively).
1
revealed by H NMR analysis of the produced acater-
in.14 Dehydroacaterin reductase was found to contain
tightly bound FAD (unpublished data). It is thus likely
that the hydride transfer from NADPH to acaterin takes
place via the flavin cofactor and this was confirmed by
spectroscopic data monitoring the reduction of FAD
(data not shown). We propose that the reactions cata-
lyzed by dehydroacaterin reductase proceed as shown
in Scheme 2. Quite recently we have completed the puri-
fication of dehydroacaterin reductase. Details of the
purification and the properties of dehydroacaterin
reductase will be reported elsewhere.
4. Experimental
4.1. General
The cell-free preparation was fractionated by
(NH4)2SO4 precipitation (30–55%) in a standard man-
ner. The precipitates were recovered by centrifugation
(20,000g, 10 min) and redissolved in a minimum amount
of 20 mM Tris–HCl buffer (pH 7.5), and dialyzed
against the same buffer for 4 h at 4 ꢁC. The solution
were applied to a DEAE column (2.5 · 25 cm). The col-
umn was eluted with a linear gradient of 0–0.5 M NaCl
in 20 mM Tris–HCl buffer (pH 7.5). The active frac-
tions, eluted with ca. 0.2 M NaCl, were combined and
concentrated to a reduced volume (3 mL) through a
membrane filter (Vivaspin 20 Polyethersulfone
VS2001) by centrifugation at (6000g, 1 h). The concen-
trated enzyme solution was volumed up to 10 mL with
0.1 M sodium phosphate buffer (pH 7.4). This enzyme
solution (2.3 mg/mL protein) was used for the study of
the utilization of the C-4 hydrogen of NADPH.
2H NMR spectra were recorded on a JEOL LA-400 or
Bruker DRX500 spectrometer using CHCl3 as a solvent.
A signal of natural abundance deuterium of the solvent
was used as internal standard (d 7.26). Protein concen-
tration was determined by the Lowry procedure with
ovalbumin as standard.
4.2. Materials
Dehydroacaterin was prepared by the fermentation of
Pseudomonas sp. A92 as described previously.6 NADP+
and NADPH were purchased from Wako Pure Chemi-
cal Industries (Osaka). [1-2H]Glucose and T. acidophi-
lum and Cryptococus uniguttulas dehydrogenases were
obtained from Sigma, Toyopearl DEAE-650M from
Tosoh (Tokyo). [4,4-2H2]NADPH was kindly provided
by Ms. K. Takahashi, Kyoritsu College of Pharmacy.
4.4. Incubation in D2O
4.3. Cell-free preparation
A portion (6.5 mL) of the 20,000g supernatant was
mixed with 0.1 M sodium phosphate buffer (pH 7.4)
(13 mL) prepared in D2O. To this solution was added
Maintenance of Pseudomonas sp. A92 and fermentation
conditions are described previously.6 In general, 48-h

6408
S. Nakano et al. / Bioorg. Med. Chem. 14 (2006) 6404–6408
NADPH (22 mg)/H2O (1.1 mL) and 2 (2.0 mg)/acetone
(0.6 mL). The mixture was incubated at 30 ꢁC for 2 h
aerobically. AcOEt (20 mL) was added and the mixture
was vortexed, and centrifuged at 1500g for 10 min to get
a clear organic layer. The AcOEt layer was set aside and
the H2O layer was re-extracted with AcOEt. The com-
bined organic layer was washed with sat. NaHCO3
and brine, dried over Na2SO4, and concentrated to dry-
ness. The residue was purified by p-TLC to give a pure
product (2.1 mg). The 2H NMR spectrum of the sample
is shown in Figure 1.
4.7. Incubation of [4-proR-2H]- and [4-proS-2H]NADPHs
To the enzyme solution after the DEAE column (10 mL,
2.3 mg/mL protein) were added [4-proR-2H]NADPH
(2.5 mg)/50 mM sodium phosphate buffer (1.0 mL) and
2 (1.0 mg)/acetone (0.1 mL). The mixture was incubated
at 30 ꢁC for 1 h and worked up as described above. The
purified product (0.6 mg) was analyzed by 2H NMR
spectroscopy (Fig. 3). [4-proS-2H]NADPH (2.5 mg)/
50 mM sodium phosphate buffer (1.0 mL) and 2
(1.0 mg)/acetone (0.1 mL) were similarly incubated with
2
the enzyme solution (10 mL) to give 1 (0.6 mg). The H
NMR spectrum of 1 is shown in Figure 3.
4.5. Incubation in the presence of [4,4-2H2]NADPH
[4,4-2H2]NADPH (64 mg)/H2O (3.2 mL) and 2 (6.7 mg)/
acetone (2.0 mL) were added to a portion (35 mL) of the
20,000g supernatant. The mixture was incubated and
worked up as described above to give acaterin (2.3 mg).
The 2H NMR spectrum of the sample is shown in
Figure 1.
Acknowledgments
We thank Professors A. Endo and H. Hosomi, Tokyo
Noko University, for providing a strain of Pseudomonus
sp. A 92. Thanks are also due to Ms. K. Takahashi,
Kyoritsu College of Pharmacy, for [4,4-2H2]NADPH.
4.6. Preparation of [4-pro-R-2H]- and [4-pro-
S-2H]NADPHs
References and notes
To a solution of NADP+ (4.6 mg) and [1-2H]glucose
(36 mg) in 50 mM sodium phosphate buffer (1.0 mL,
pH 7.0) was added a glucose dehydrogenase solution
from T. acidophilum Recombinant (2.5 lL, 9.9 mg pro-
tein/mL). The mixture was incubated at 37 ꢁC for 1 h.
The reaction mixture was centrifuged at 6000g for 1 h
using a membrane filter to yield the filtrate (1 mL) con-
taining 2H-labeled NADPH (2.5 mg, determined by UV
measurement). This was used for incubation without
further purification.
1. Naganuma, S.; Sakai, K.; Hasumi, K.; Endo, A. J.
Antibiot. 1992, 45, 1216–1221.
2. Sekiyama, Y.; Araya, H.; Hasumi, K.; Endo, A.; Fujim-
oto, Y. Nat. Prod. Lett. 1997, 11, 61–67.
3. Pattenden, G. Prog. Chem. Org. Nat. Prod. 1978, 35, 133–
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He, K.; McLaughlin, J. L. Nat. Prod. Rep. 1996, 13, 275–
306.
4. Sekiyama, Y.; Araya, H.; Hasumi, K.; Endo, A.; Fujim-
oto, Y. Tetrahedron Lett. 1998, 39, 6233–6236.
5. Sekiyama, Y.; Araya, H.; Hasumi, K.; Endo, A.; Fujim-
oto, Y. Tetrahedron Lett. 1999, 40, 4223–4226.
6. Sekiyama, Y.; Fujimoto, Y.; Hasumi, K.; Endo, A. J. Org.
Chem. 2001, 66, 5649–5654.
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In the other run, the filtrate was further purified in order
to record the 1H NMR spectrum. The filtrate was applied
to a DEAE column (1 · 10 cm) and the column was eluted
with an increasing concentration of NH4HCO3 from 0 to
0.5 M in 0.1 M potassium phosphate buffer (1.0 mL, pH
7.0). The fractions containing NADPH (eluted at ca.
0.2 M) were combined and freeze-dried. The resulting sol-
id was dissolved in distilled water (0.5 mL) and diluted
with methanol (5 mL) to cause precipitation of most of
the inorganic salt. The methanol layer was taken up and
concentrated to give a purified [4-pro-S-2H]NADPH.
The sample was analyzed by 1H NMR spectroscopy
(Fig. 2). By replacing the glucose dehydrogenase with
Cryptcocus uniguttulatus glucose dehydrogenase
(0.5 mg) (pH 8.0, sodium phosphate buffer was used in-
stead of pH 7.0), [4-pro-S-2H]NADPH (2.5 mg) was ob-
tained from 4.6 mg NADP+. The 1H NMR spectrum of
the labeled sample purified by a DEAE column as de-
scribed above is shown in Figure 2.
14. The proton signal at C-5 of 1 was observed as 2.15 units of
hydrogen (theoretically 2.0 units), thus indicating that
85% of 4-pro-R-2H was transferred to the C-5 position.