K. Ichinose et al.
The known examples of two-component FMOs catalyze only
Preparation of recombinant proteins and substrates for enzyme
assays: Construction of the E. coli expression plasmids for ActVA-
ORF5 and ActVB, culture conditions, and the procedure for purifi-
cation of recombinant ActVA-ORF5 (His-ActVA-ORF5) and ActVB
[
4]
a limited range of reactions. Together with our previous find-
[
13]
ing of the quinone-forming activity, this study has demon-
strated an additional in vitro epoxidation activity of the ActVA-
ORF5/ActVB system. This additional function is possibly a result
of the adaptable structural features of the two-component
system, in which a flavin reduction and a substrate oxygena-
tion are catalyzed by separate proteins. The substrate mole-
cules (either DDHK or KAL) would be acted on in the catalytic
pocket of the oxygenase component, ActVA-ORF5, in which
the distinct reactions would proceed on the corresponding re-
active carbons. Further characterization of this enzyme system,
particularly for the exact functions of ActVA-ORF5 and ActVB in
an epoxyquinone formation, is in progress.
[13]
(His-ActVB) have been described previously.
His-ActVA-ORF5
(129.6 mg) and His-ActVB (6.5 mg) were prepared. Culturing of
S. tanashiensis and isolation of DHK were also carried out as previ-
[13]
13
ously described. Details of the feeding experiment with [ C ]-
2
acetic acid are described in the Supporting Information. For the
preparation of KAL from DHK, the compound was dissolved in eth-
ylene glycol monomethyl ether (EGME, 0.17 mm) and kept over-
night at 48C. Boiled enzyme proteins for control experiments were
prepared by immersion in boiling water for 5 min.
In vitro enzyme assay: The assay mixture (500 mL) contained
NADH (500 mm), FMN (5 mm), EGME (30%, v/v), catalase
Another epoxyquinone derivative of a BIQ, NNM-E, has been
À1
(
120 UmL ), His-ActVB (100 nm), His-ActVA-ORF5 (1 mm), and a
[
14]
characterized from Streptomyces rosa var. notoensis. Further-
more, the NNM-A monooxygenase (epoxidizing) activity was
demonstrated in a cell-free system in the presence of NAD(P)H
and molecular oxygen. Interestingly, this epoxidation reaction
did not occur with other NNM-A derivatives or with deoxyfre-
nolicin, which possess the opposite stereochemical configura-
tion to NNMs as well as a different chain length, thus indicat-
ing relatively rigid substrate recognition by this enzyme. A
notable difference between the NNM-A monooxygenase and
the ActVA-ORF5/ActVB system is a strict structural requirement
for the C-1 dihydro (carboxylic acid) form, rather than the C-1
lactone form, for the epoxidation reaction catalyzed by the
former enzyme.
substrate [DHK, KAL, or NNM_D (50 mm)] in PIPES-NaOH (25 mm,
pH 7.0). After the mixture had been incubated at 258C for a suita-
ble period, an aliquot of the assay mixture solution (50 mL) was di-
rectly subjected to HPLC analysis under the following conditions:
column, TSK gel ODS-100S (4.6 mm i.d.ꢁ150 mm, TOSOH); column
temperature, 408C; gradient elution, solvent A (0.5% acetic acid in
CH CN) and solvent B (0.5% acetic acid in deionized H O); gradient
3
2
profile: 0–5 min, 35% A; 5–20 min: 35–71% A; flow rate,
À1
1
.0 mLmin ; detection, absorption between 250 and 600 nm with
a photo-diode array detector (TOSOH PD-8020).
Large-scale enzymatic preparation of epo-KALs: The reaction
mixture (5 mL) contained NADH (1.0 mm), FMN (10 mm), EGME
À1
(
30%, v/v), catalase (120 UmL ), His-ActVB (100 nm), His-ActVA-
ORF5 (1 mm), and the substrate (KAL, 50 mm) in PIPES/NaOH
25 mm, pH 7.0). After incubation at 258C for 30 min, the reaction
mixture was acidified with HCl (1m) and centrifuged at 9000g for
0 min to remove precipitate. The reaction products were then ex-
Although no epoxide intermediate or shunt metabolite relat-
ed to ACT biosynthesis has been characterized from S. coelico-
lor A3(2), this finding of an additional epoxidation activity of
the two-component FMO ActVA-ORF5/ActVB is potentially val-
uable for further understanding of the later tailoring steps in
the biosynthesis of BIQ antibiotics.
(
1
tracted with an equal volume of ethyl acetate twice. Ethyl acetate
was removed in vacuo, and the resulting EGME solution was
applied to a SepPak cartridge (Waters). After the resin had been
washed with dH O, epo-KALs were eluted with methanol and fur-
2
13
ther purified by preparative HPLC as described above. C-labeled
KAL (19.8 mg) was subjected to 248 assays to give (5S,14R)-epo-
KAL (X, 1.9 mg) and (5R,14S)-epo-KAL (Y, 3.7 mg) for NMR spectros-
copy. Unlabeled (5S,14R)-epo-KAL (3.2 mg) was prepared from 362
assays with unlabeled KAL (27.7 mg) for X-ray crystallography.
Experimental Section
General: LC/ESI-TOF-MS analysis was carried out with a Waters Alli-
anceHT instrument fitted with a photo-diode array detector
(
Waters 2995). HPLC analysis was performed with a TOSOH SC8020
Chemical conversion of DHK into epo-KALs: DHK was dissolved
in pyridine (1 mgmL ), and the mixture was allowed to stand at
À1
system and photo-diode array detector. Mass spectrometry data
were obtained by electron spray ionization (ESI) with a Waters LCT-
Premier instrument. Optical rotations were measured with a Jasco
P-1010 digital polarimeter. NMR spectra were obtained with a JEOL
ECX400 spectrometer (400 MHz for H; 100 MHz for C) in
[
RT. An aliquot (5 mL) was directly subjected to HPLC analysis under
the same conditions as described for the in vitro enzyme assay,
except for the column (TSK gel ODS-100Z, 4.6 mm i.d.ꢁ150 mm,
TOSOH). The reaction products were confirmed to be identical to
those obtained from the corresponding enzymatic reactions by
spectroscopic analysis.
1
13
1
13
D ]acetone. H and C chemical shifts were referenced to the sol-
6
vent signals (d =2.04 ppm and d =29.8 ppm). Melting points
H
C
were determined with a Yanagimoto micro melting point appara-
tus and were uncorrected.
Structure elucidation of the products: By LC/HR-ESI-MS analysis,
the molecular formulas of both X and Y were determined to be
Chemicals: All chemicals were reagent grade purchased from
À
C H O . (Calcd. for X: 315.0505 [MÀH] ; found: 315.0504; calcd.
1
6
12
7
Wako Pure Chemical Industries or Sigma–Aldrich unless otherwise
À
1
3
13
for Y: 315.0505 [MÀH] ; found: 315.0513). Identical UV/Vis spectra
stated. [ C ]Acetic acid (99% atom C) was purchased from Isotec
2
were obtained for X and Y, as shown in Figure 1B. Optical rotations
Co., Ltd. (Miamisburg, OH, USA). Nanaomycin D was kindly provid-
ed by Prof. Heinz G. Floss, University of Washington, Seattle, USA.
for X and Y were [a] =+10.08 (c=0.1 in MeOH) and +45.38 (c=
D
0
.1 in MeOH), respectively. The IUPAC names of the elucidated
Bacterial strains: Escherichia coli BL21(DE3)/pLysS was used as the
host for protein expression. Streptomyces tanashiensis JCM4086
structures of (5S,14R)-epo-KAL and (5R,14S)-epo-KAL are (5aS,11aR)-
epoxy-3,3aR,5R,11bR-tetrahydro-7-hydroxy-5-methyl-2H-furo[3,2-b]-
(
RIKEN) was used for the isolation of DHK.
naphtho[2,3-d]pyran-2,6,11-trione
and
(5aR,11aS)-epoxy-
2
772
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 2767 – 2773