A biosynthetic pathway for BE-7585A, a 2-thiosugar-containing angucycline-type natural product

Article abstract of DOI:10.1021/ja1014037

Sulfur is an essential element found ubiquitously in living systems. However, there exist only a few sulfur-containing sugars in nature and their biosyntheses have not been studied. BE-7585A produced by Amycolatopsis orientalis subsp. vinearia BA-07585 has a 2-thiosugar and is a member of the angucycline class of compounds. We report herein the results of our initial efforts to study the biosynthesis of BE-7585A. Spectroscopic analyses verified the structure of BE-7585A, which is closely related to rhodonocardin A. Feeding experiments using ¹³C-labeled acetate were carried out to confirm that the angucycline core is indeed polyketide-derived. The results indicated an unusual manner of angular tetracyclic ring construction, perhaps via a Baeyer-Villiger type rearrangement. Subsequent cloning and sequencing led to the identification of the bex gene cluster spanning ～30 kbp. A total of 28 open reading frames, which are likely involved in BE-7585A formation, were identified in the cluster. In view of the presence of a homologue of a thiazole synthase gene (thiG), bexX, in the bex cluster, the mechanism of sulfur incorporation into the 2-thiosugar moiety could resemble that found in thiamin biosynthesis. A glycosyltransferase homologue, BexG2, was heterologously expressed in Escherichia coli. The purified enzyme successfully catalyzed the coupling of 2-thioglucose 6-phoshate and UDP-glucose to produce 2-thiotrehalose 6-phosphate, which is the precursor of the disaccharide unit in BE-7585A. On the basis of these genetic and biochemical experiments, a biosynthetic pathway for BE-7585A can now be proposed. The combined results set the stage for future biochemical studies of 2-thiosugar biosynthesis and BE-7585A assembly.

Full text of DOI:10.1021/ja1014037

Published on Web 05/05/2010
A Biosynthetic Pathway for BE-7585A, a
2-Thiosugar-Containing Angucycline-Type Natural Product
Eita Sasaki, Yasushi Ogasawara, and Hung-wen Liu*
DiVision of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and
Biochemistry, UniVersity of Texas at Austin, Austin, Texas 78712
Received February 17, 2010; E-mail: h.w.liu@mail.utexas.edu
Abstract: Sulfur is an essential element found ubiquitously in living systems. However, there exist only a
few sulfur-containing sugars in nature and their biosyntheses have not been studied. BE-7585A produced
by Amycolatopsis orientalis subsp. vinearia BA-07585 has a 2-thiosugar and is a member of the angucycline
class of compounds. We report herein the results of our initial efforts to study the biosynthesis of BE-
7
585A. Spectroscopic analyses verified the structure of BE-7585A, which is closely related to rhodonocardin
13
A. Feeding experiments using C-labeled acetate were carried out to confirm that the angucycline core is
indeed polyketide-derived. The results indicated an unusual manner of angular tetracyclic ring construction,
perhaps via a Baeyer-Villiger type rearrangement. Subsequent cloning and sequencing led to the
identification of the bex gene cluster spanning ∼30 kbp. A total of 28 open reading frames, which are likely
involved in BE-7585A formation, were identified in the cluster. In view of the presence of a homologue of
a thiazole synthase gene (thiG), bexX, in the bex cluster, the mechanism of sulfur incorporation into the
2-thiosugar moiety could resemble that found in thiamin biosynthesis. A glycosyltransferase homologue,
BexG2, was heterologously expressed in Escherichia coli. The purified enzyme successfully catalyzed the
coupling of 2-thioglucose 6-phoshate and UDP-glucose to produce 2-thiotrehalose 6-phosphate, which is
the precursor of the disaccharide unit in BE-7585A. On the basis of these genetic and biochemical
experiments, a biosynthetic pathway for BE-7585A can now be proposed. The combined results set the
stage for future biochemical studies of 2-thiosugar biosynthesis and BE-7585A assembly.
Introduction
persulfide, protein thiocarboxylate, or S-adenosylmethionine-
dependent radical are often generated in the enzymes carrying
The angucycline class of natural products, which have a
characteristic fused four-ring frame assembled in an angular
manner, are rich in antibacterial or anticancer activities. BE-
out such reactions. Whether the same or different mechanisms
are used to form the C-S bond in 2-thiosugar biosynthesis is
the focus of this study.
1
7
585A (1), produced by Amycolatopsis orientalis subsp. Vinearia
Thus far, only a handful of thiosugar-containing natural
BA-07585, is an angucycline-type natural product that inhibits
thymidylate synthase. In addition to the benz[a]anthraquinone
2
2
products are known. These include BE-7585A (1), rhodono-
4
5
6
7
cardins (5), lincomycins (6), celesticetin, calicheamicins (7),
core, BE-7585A also contains the deoxysugar, rhodinose (2),
and a disaccharide appendage (4) containing a highly unusual
8
9
10
11
14
esperamicins, namenamicin, shishijimicins, albomycins (8)
and SB-217452 from microorganisms; kotalanol, salacinol,
12
13
2
-thioglucose (3). While sulfur is an essential element found
ubiquitously in living systems, including amino acids, nucleic
acids, metal clusters, enzyme cofactors, and many secondary
metabolites, it is seldom found in carbohydrates. The rareness
of the thiosugar entity prompted us to explore the biosynthesis
of BE-7585A, especially the mode of sulfur incorporation into
the 2-thiosugar moiety (3). Mechanistic details of several
biological sulfur insertion reactions have recently been unrav-
(
(
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10.1021/ja1014037  2010 American Chemical Society
J. AM. CHEM. SOC. 2010, 132, 7405–7417 9 7405

A R T I C L E S
Sasaki et al.
Interestingly, feeding experiments using 13C-labeled acetate
suggest that the benz[a]anthraquinone core formation may
involve an oxidative ring cleavage followed by rearrangement
and recyclization leading to the observed angular-tetracyclic
structure. Subsequent cloning and sequencing experiments led
to the identification of the bex gene cluster from A. orientalis
spanning ∼30 kbp. On the basis of the functional prediction of
the genes in the cluster, a possible biosynthetic pathway
culminating in 1 is proposed. The bexX gene, which encodes a
protein homologous to thiazole synthase (ThiG), likely plays a
key role in sulfur incorporation during 2-thiosugar formation.
The in vitro characterization of BexG2, which catalyzes the
coupling of 2-thioglucose 6-phosphate and UDP-glucose to
produce the precursor of the disaccharide unit (4) in 1 is also
reported. Overall, this work provides significant insights into
the biosynthesis of the 2-thiosugar and the unusual ring
formation of the benz[a]anthraquinone core in BE-7585A.
Elucidation of unusual sugar biosynthesis is important not only
for understanding the chemical mechanisms of their formation,
but also for future pathway engineering experiments to construct
Chart 1. Structures of Compounds 1-8
“
tailor-made” glycosylated metabolites with new or enhanced
2
0
biological activities.
Experimental Procedures
Materials. All chemicals and reagents were purchased from
Sigma-Aldrich Chemical Co. (St. Louis, MO) or Fisher Scientific
(Pittsburgh, PA), and were used without further purification unless
otherwise specified. Enzymes and molecular weight standards used
for the cloning experiments were products of Invitrogen (Carlsbad,
CA) or New England Biolabs (Ipswich, MA). Ni-NTA agarose and
kits for DNA gel extraction and spin minipreps were obtained from
Qiagen (Valencia, CA). PfuUltra DNA polymerase and Gigapack
III XL packaging extract were products of Stratagene (La Jolla,
CA). Growth medium components were acquired from Becton
Dickinson (Sparks, MD). Reagents for sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) were purchased
from Bio-Rad (Hercules, CA), with the exception of the protein
molecular weight markers, which were obtained from Invitrogen
or New England Biolabs. Sterile syringe filters were products of
Fisher Scientific. Amicon YM-10 ultrafiltration membranes were
purchased from Millipore (Billerica, MA). The CarboPac PA1 high-
performance liquid chromatography (HPLC) column was obtained
from Dionex (Sunnyvale, CA). Oligonucleotide primers were prepared
by Invitrogen or Integrated DNA Technologies (Coralville, IA).
Bacterial Strains and Plasmids. A. orientalis subsp. Vinearia
BA-07585 was generously provided by Banyu Pharmaceutical Co.
1
5
16
and glucosinolates from plants; and 5-thio-D-mannose from
the marine sponge Clathria pyramida (although the actual
producer might be its symbiont). (The structures of compounds
1
-8 are shown in Chart 1.) The biosynthetic gene clusters for
some of the compounds produced by bacteria have been
17-19
identified,
but the pathway and mechanisms of the thiosugar
biosynthesis in each case remain obscure. Among these
compounds, BE-7585A (1) and rhodonocardins (5) are the only
two reported natural products containing a 2-thiosugar (3).
Interestingly, 1 and 5 are constitutional isomers that share the
benz[a]anthraquinone core structure and three sugar components,
rhodinose, 2-thioglucose, and glucose, but differ in the attach-
ment of the 2-thioglucose and glucose moieties on the core
molecule. In BE-7585A (1), the 2-thioglucose and glucose
moieties are linked via their anomeric carbons, and the resulting
disaccharide (4) is attached to the C-5 position of the aglycone
through a thioether linkage. In contrast, the 2-thioglucose and
glucose in 5 are separately attached to C-5 and C-4a of the
angucycline core via O-glycosidic bonds, leaving the 2-mer-
capto-substituent in 3 as a free thiol group. The structure of
(
Tokyo, Japan). Escherichia coli DH5R, acquired from Bethesda
Research Laboratories (Gaithersburg, MD), was used for routine
cloning experiments. E. coli XL-1 Blue MRF′, purchased from
Stratagene, was employed for cosmid manipulations. Protein
overexpression host E. coli BL21 star (DE3) was obtained from
Invitrogen. Plasmids pGEM-T Easy and pUC119, used for sub-
cloning, were products of Promega (Madison, WI) and Takara Bio
4
rhodonocardin (5) is fully documented, but that of BE-7585A
1) is not because the structure is reported in a patent.
Here, we verified the structure of BE-7585A (1) and confirm
that the angucycline core of 1 is indeed polyketide-derived.
2
(
(
Shiga, Japan), respectively. Vector pET28b(+) for protein over-
(
(
(
(
(
13) Yoshikawa, M.; Murakami, T.; Yashiro, K.; Matsuda, H. Chem. Pharm.
expression was purchased from Novagen (Madison, WI).
General. NMR spectra were acquired on a Varian Unity 500
MHz spectrometer, and chemical shifts (in ppm) are reported
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relative to that of the solvent peak (δ
) 3.30 and δ ) 49.0 for deuterated methanol) or an internal
standard (dioxane, δ ) 67.4) for spectra taken in D O solvent.
H
) 4.65 for deuterated water,
15) Gershenzon, J.; Halkier, B. A. Annu. ReV. Plant Biol. 2006, 57, 303–
3
33.
δ
H
C
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C
2
1
17) Ahlert, J.; Shepard, E.; Lomovskaya, N.; Zazopoulos, E.; Staffa, A.;
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(
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19) Eerm a´ k, L.; Novotn a´ , J.; S a´ gov a´ -Mare e` kov a´ , M.; Kopeck y´ , J.;
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7
406 J. AM. CHEM. SOC. 9 VOL. 132, NO. 21, 2010

Biosynthesis of BE-7585A
A R T I C L E S
a
HPLC was performed on a Beckman Coulter System Gold equipped
with a UV detector or Corona CAD (charged aerosol detector).
DNA sequencing was performed by the core facility of the Institute
of Cellular and Molecular Biology at the University of Texas,
Austin. Vector NTI Advance 10.1.1 from Invitrogen was used for
sequence alignments. Mass spectra were recorded by the Mass
Spectrometry core facility in the Department of Chemistry and
Biochemistry at the University of Texas, Austin. Standard genetic
manipulations of E. coli were performed as described by Sambrook
Table 1. Primers Used for Cosmid Library Screening
primer name sequence
KS
KS
R
-forward
-reverse
5′-CGACGCVCCSATCDCVCCSATC-3′
5′-GGAANCCDCCGAABCCGCTGCC-3′
5′-AGCTSTCSCCSACVGTBCAGC-3′
5′-WRGAAVCGRCCSCCYTCYTCSG-3′
5′-TSAACCCGMTCVTSCAGACGG-3′
5′-CSGGRTGSCKGGTSAKGTTSCC-3′
R
2,3-dehydratase-forward
2,3-dehydratase-reverse
3-dehydrase-forward
3-dehydrase-reverse
2
1
a
et al.
M ) AC, R ) AG, W ) AT, S ) CG, Y ) CT, K ) GT, V )
ACG, H ) ACT, D ) AGT, B ) CGT, N ) ACGT.
Production, Isolation, and Identification of BE-7585A (1).
Spores of A. orientalis subsp. Vinearia BA-07585 were inoculated
into 100 mL of International Streptomyces Project (ISP) medium
22
BamHI site of the pOJ446 vector. The ligated DNA was packaged
into phage particles using a Gigapack III XL packaging extract
and then introduced into E. coli XL1-Blue MRF′ according to the
manufacturer’s instructions. Recombinants were selected on
Luria-Bertani (LB)-agar plates in the presence of apramycin (50
µg/mL). After incubation for 16 h, the resulting transformants were
spotted within grids on new plates. This genomic library contains
a total of 454 cosmids.
2
and grown in a rotary incubator at 29 °C and 250 rpm for 5
days. The resultant seed culture (25 mL) was transferred to 1 L of
ISP-2 medium and grown under the same conditions for 10 days.
The growth culture was centrifuged at 6000g for 10 min to remove
the cells and other insoluble materials. The supernatant was applied
to a synthetic polymeric adsorbent Diaion HP20 column (250 mL)
and washed with water. The adsorbed compounds were then eluted
with 500 mL of methanol. The collected red fractions were pooled
and concentrated by rotary evaporation in vacuo and further purified
on a Diaion CHP20P column (50 mL) using 5-20% of methanol
in water as the eluent. The typical yield was 100-150 mg of BE-
PCR-Based Screening of the Cosmid Library. Polymerase
chain reaction (PCR) primers for screening of type II PKS were
designed based on multiple sequence alignments of 10 known
ꢀ
-ketoacyl synthases R subunit (KSR) of actinomycetes including
23 24
Streptomyces Venezuelae and Streptomyces coelicolor A3(2),
7
585A (1) from 1-L of culture. The structure of 1 was determined
1 13
which are available in the National Center for Biotechnology
Information (NCBI) database. Similarly, the primers for deoxysugar
by H, C, COSY, HSQC, HMBC, and NOESY NMR experiments
and ESI-MS.
2
5
13C-Labeling Experiments. Spores of A. orientalis subsp.
genes, such as TDP-6-deoxy-4-keto-D-glucose 2,3-dehydratase
and TDP-2,6-dideoxy-4-keto-D-glucose 3-dehydrase, were de-
signed based on the reported gene sequences from eight actino-
26
Vinearia BA-07585 were inoculated into 50 mL of ISP-2 medium
and grown in a rotary incubator at 30 °C and 250 rpm for 5 days.
The resultant seed culture (25 mL) was transferred to 1 L of ISP-2
medium and grown under the same conditions for 2 days. Sodium
2
7
mycete strains including Streptomyces fradiae and Streptomyces
2
8
cyanogenus. The sequences of the primers used for the PCR-
based screening are listed in Table 1. PCR was performed using
PfuUltra DNA polymerase with these primers and the cosmid DNA
from the genomic library. Amplified DNA fragments were ligated
into the pGEM-T Easy vector for cloning and sequencing.
Sequencing and Gene Cluster Identification. Cosmids A108
and C006, which together span the entire bex cluster, were digested
with either KpnI, NcoI, or PstI. The resulting 2.5-12 kb DNA
fragments were subcloned into a modified pUC119 vector, whose
1
3
[
1- C]acetate, in an aqueous solution (4%, 5 mL) sterilized by
filtration through a syringe filter (0.2 µm), was added to the culture,
13
which had a light red color. An additional [1- C]acetate solution
was added (4%, 5 mL × 4) to the growth culture every 13-20 h.
The final concentration of labeled acetate was 0.1%. After a 10-
day incubation period, the culture was centrifuged at 6000g for 10
min. The 13C-labeled BE-7585A (1, 150 mg) was isolated from
the supernatant by using Diaion HP20 and CHP20P columns as
2
9
multiple cloning site harbors extra restriction sites. Both strands
of the subclones were sequenced using M13 universal primers or
by primer walking. Sequencing data were obtained using a capillary-
based AB 3700 DNA analyzer and assembled using Vector NTI
Suite program. Open reading frame assignments were made with
described above for the purification of the nonlabeled 1. The purified
compound was analyzed by 13C NMR spectroscopy.
The feeding experiment was also performed with sodium [1,2-
13C
]acetate in 1-L ISP-2 medium. A 25 mL aqueous solution
2
13
containing both sodium [1,2- C
2
]acetate (0.68%) and nonlabeled
30
the assistance of FramePlot 2.3.2. Homologous protein sequences
were identified in the NCBI database using the basic local alignment
search tool (BLAST).
sodium acetate (1.3%) was prepared and sterilized by filtration
through a syringe filter. The solution (10 mL) was then added to
the culture, which had been grown for 2 days and turned to light
red. The remaining solution (15 mL) was added 20 h after the first
addition. The final concentration of the labeled acetate was 0.017%.
After a 10-day incubation period, the culture was centrifuged at
(
(
22) Bierman, M.; Logan, R.; O’Brien, K.; Seno, E. T.; Nagaraja Rao, R.;
Schoner, B. E. Gene 1992, 116, 43–49.
23) Han, L.; Yang, K.; Ramalingam, E.; Mosher, R. H.; Vining, L. C.
Microbiology 1994, 140, 3379–3389.
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inson, C. R.; Butler, M. J.; Sugden, D. A.; Warren, M.; McKillop, C.;
Bailey, C. R.; Humphreys, G. O.; Hopwood, D. A. Nature 1987, 325,
6
000g for 10 min to remove cells and any insoluble materials. The
C-labeled BE-7585A (1, 80 mg) was isolated from the supernatant
13
1
3
using Diaion HP20 and CHP20P columns and analyzed by
NMR spectroscopy as described above.
C
Construction of the Cosmid Library. Spores of A. orientalis
subsp. Vinearia BA-07585 were inoculated into 50 mL of tryptone
soya broth (TSB) medium and grown in a rotary incubator at 30
8
18–821.
(
25) (a) Chen, H.; Agnihotri, G.; Guo, Z.; Que, N. L. S.; Chen, X.; Liu,
H.-w. J. Am. Chem. Soc. 1999, 121, 8124–8125. (b) Draeger, G.; Park,
S.-H.; Floss, H. G. J. Am. Chem. Soc. 1999, 121, 2611–2612. (c)
Zhang, H.; White-Phillip, J. A.; Melancon, C. E., III.; Kwon, H.-j.;
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26) Hong, L.; Zhao, Z.; Liu, H.-w. J. Am. Chem. Soc. 2006, 128, 14262–
°C and 250 rpm for 3 days. The mycelia were harvested by
centrifugation at 5000g for 30 min. The cells were disrupted by
lysozyme and SDS, and the cell lysate was treated with proteinase
K. The released genomic DNA was isolated by phenol-chloroform
extraction followed by sodium acetate-isopropyl alcohol precipita-
tion. The DNA was partially digested with Sau3AI restriction
endonuclease in a time dependent manner, and ligated into the
(
(
1
4263.
27) Hoffmeister, D.; Ichinose, K.; Domann, S.; Faust, B.; Trefzer, A.;
Dr a¨ ger, G.; Kirschning, A.; Fischer, C.; K u¨ nzel, E.; Bearden, D. W.;
Rohr, J.; Bechthold, A. Chem. Biol. 2000, 7, 821–831.
(
28) Westrich, L.; Domann, S.; Faust, B.; Bedford, D.; Hopwood, D. A.;
Bechthold, A. FEMS Microbiol. Lett. 1999, 381–387.
(
21) Sambrook, J.; Russell, D. W. Molecular Cloning: A Laboratory
Mannual, 3rd ed.; Cold Spring Harbor Laboratory Press:: Cold Spring
Harbor, NY, 2001.
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J. AM. CHEM. SOC. 9 VOL. 132, NO. 21, 2010 7407

A R T I C L E S
Sasaki et al.
Cloning of bexG2. The bexG2 gene was PCR-amplified from
cosmid C006 using primers with engineered NdeI and HindIII
restriction sites. The sequences of the primers are 5′-GGTTAG-
GCATATGAGGATTCTGACCTGCAGC-3′ (forward) and 5′-
GTAGAAGCTTAGGTCAGAGCCCGAAAA-CCCC-3′ (reverse).
The engineered restriction sites are shown in bold, the start codon
in bold and also underlined, and the stop codon underlined. The
PCR-amplified gene fragments were purified, digested with NdeI
and HindIII, and ligated into pET28b(+) vector digested with the
same enzymes. The resulting plasmid, bexG2/pET28b(+), was used
to transform E. coli BL21 star (DE3) strain for protein overex-
pression. The BexG2 enzyme was expressed as an N-terminal-His
6
-
tagged protein.
Expression and Purification of BexG2. An overnight culture
of E. coli BL21 star (DE3)-bexG2/pET28b(+), grown in the LB
medium (10 mL) containing 50 µg/mL of kanamycin at 37 °C,
was used to inoculate 1 L of the same growth medium. The culture
was incubated at 37 °C with shaking (230 rpm) until the OD600
reached ∼0.5. Protein expression was then induced by the addition
of isopropyl ꢀ-D-1-thiogalactopyranoside (IPTG) to a final con-
centration of 0.1 mM, and the cells were allowed to grow at 18 °C
and 125 rpm for an additional 24 h. The cells were harvested by
centrifugation at 4500g for 15 min and stored at -80 °C until lysis.
All purification steps were carried out at 4 °C using Ni-NTA resin
according to the manufacturer’s protocol with minor modifications.
Specifically, the thawed cells (3 g) were resuspended in the lysis
buffer (15 mL) containing 10% (v/v) glycerol and 10 mM imidazole.
After incubation with lysozyme (15 mg) for 30 min, the cells were
disrupted by sonication using 10 × 10-s pulses with a 30-s cooling
pause between each pulse. The resulting lysate was centrifuged at
Figure 1. SDS-PAGE gel of the purified N-His
6
tagged BexG2 (482 aa,
3.6 kDa). The molecular weight marks are 220, 160, 120, 100, 90, 80, 70,
0, 50, 40, 30, 25, 20, 15, and 10 kDa (top to bottom).
5
6
2
. 1,3,4,6-Tetra-O-acetyl-2-O-trifluoromethylsulfonyl-ꢀ-D-
mannopyranose (12). Compound 12 was synthesized from 11 using
a previously reported procedure with minor modifications. Specif-
ically, to a solution of 11 (3.5 g, 10 mmol) in anhydrous methylene
chloride (100 mL) was added anhydrous pyridine (1.8 mL, 22
mmol). The solution was cooled to -20 °C and treated with
trifluoromethane-sulfonic anhydride (3.6 mL, 22 mmol), which was
added dropwise in 1 h under N . The resulting suspension was
2
stirred at room temperature for 1 h. The mixture was washed
sequentially with cold water, saturated sodium bicarbonate, and
1
0 000g for 20 min, and the supernatant was subjected to Ni-NTA
chromatography. Bound protein was eluted using buffer containing
0% glycerol and 250 mM imidazole. The collected protein solution
33
1
was dialyzed against 3 × 1-L of 50 mM Tris ·HCl buffer (pH 8)
containing 300 mM NaCl and 15% glycerol. The protein solution
was then flash-frozen in liquid nitrogen and stored at -80 °C until
31
use. Protein concentration was determined by the Bradford assay
using bovine serum albumin as the standard. The yield of BexG2
was 40 mg from 1 L culture. The molecular mass and purity of
BexG2 were estimated by SDS-PAGE analysis (Figure 1).
Synthesis of 2-Thio-D-glucose (3). 1. 1,3,4,6-Tetra-O-acetyl-
2
water; dried over anhydrous Na SO4,; and concentrated in vacuo.
The product 12 was obtained as a pale yellow solid (4.6 g, 9.6
1
mmol, 96%). The H NMR spectral data of 12 matches those
ꢀ
-D-mannopyranose (11). As shown in Scheme 1, compound 11
33b
reported previously.
was synthesized from D-mannose (9, 16.5 g, 92 mmol) following
3
2
3. 1,2,3,4,6-Penta-O,S,O,O,O-acetyl-2-thio-ꢀ-D-glucopyra-
nose (13). Compound 13 was prepared from 12 following a reported
a previously reported procedure with slight modifications. Ac-
cordingly, to a small portion of 9 (∼30 mg) in acetic anhydride
3
4
procedure with minor modifications. Accordingly, to a solution
of 12 (4.1 g, 8.5 mmol) in anhydrous dimethylformamide (170 mL)
was added potassium thioacetate (9.7 g, 85 mmol). The resulting
(
9
63 mL) was added 70% perchloric acid (5 drops). The remaining
was slowly added to the reaction mixture over a 20-min period,
keeping the reaction temperature at 40-45 °C. The resulting mixture
was stirred at room temperature for 1 h and then cooled to 15 °C.
Phosphorus tribromide (13 mL) was added dropwise to the reaction
with the reaction temperature kept at 20-25 °C. Cold water (8.3
mL) was slowly added to the resulting mixture, and the reaction
was kept at room temperature for 1.5 h. After cooling to 10 °C, a
chilled solution of sodium acetate trihydrate (50 g, 370 mmol) in
water was slowly added with the temperature kept at 25-30 °C.
The reaction mixture was stirred at room temperature for 20 min
and poured onto ice. The resulting mixture was extracted with
methylene chloride (100 mL × 2) and washed sequentially with
cold saturated sodium bicarbonate and cold water. The solution
2
mixture was stirred at room temperature for 1 h under N . After
evaporation of the solvent in vacuo, the residue was mixed with
methylene chloride and water. The organic layer was separated and
washed sequentially with water and saturated sodium chloride, dried
over anhydrous Na
was purified by silica gel column chromatography (hexanes-EtOAc,
:1) and recrystallized from hexanes-EtOAc. The product 13 was
2
SO4,, and concentrated in vacuo. The residue
2
1
obtained as a white powder (3.0 g, 7.4 mmol, 87%). The H NMR
spectrum of 13 is consistent with the literature report.
3
4a
4
. 2-Thio-D-glucose (3). To a solution of 13 (510 mg, 1.25
mmol) in anhydrous methanol (15 mL) was added Dowex 50W
X8 (∼200 mg). The reaction was gently refluxed for 15 h. The
resulting solution was filtered and the filtrate was concentrated under
reduced pressure to give an R/ꢀ mixture of 1-O-methyl-2-thio-D-
2 4
was dried over anhydrous Na SO and evaporated in vacuo. The
obtained crude mixture, which contained 2,3,4,6-tetra-O-acetyl-R-
D-mannopyranose (10) byproduct, was thoroughly washed with
anhydrous diethyl ether to give the desired product 11 as a white
1
glucopyranose (R/ꢀ ) 1:3, determined by H NMR spectroscopy).
1
The mixture was dissolved in water (20 mL) and stirred at 80 °C
powder (6.7 g, 19 mmol, 21%). The H NMR spectral data of 11
3
2b
is consistent with those previously reported.
(
33) (a) Hamacher, K. Carbohydr. Res. 1984, 128, 291–295. (b) Pavliak,
V.; Kov a´ e` , P. Carbohydr. Res. 1991, 210, 333–337.
(
(
31) Bradford, M. M. Anal. Biochem. 1976, 72, 248–254.
32) (a) Deferrari, J. O.; Gros, E. G.; Mastronardi, I. O. Carbohydr. Res.
(34) (a) Kirk, B. A.; Knapp, S. Tetrahedron Lett. 2003, 44, 7601–7605.
(b) Knapp, S.; Naughton, A. B. J.; Jaramillo, C.; Pipik, B. J. Org.
Chem. 1992, 57, 7328–7334.
1
967, 4, 432–434. (b) Kov a´ e` , P. Carbohydr. Res. 1986, 153, 168–
70.
1
7
408 J. AM. CHEM. SOC. 9 VOL. 132, NO. 21, 2010

Biosynthesis of BE-7585A
A R T I C L E S
Scheme 1
Scheme 2
4
M NH OAc (solvent B). The gradient was run from 30 to 50% B
over 10 min, 50-80% B over 5 min, with a 5 min wash at 80% B,
and 80-30% B over 5 min, followed by re-equilibration at 30% B
for 10 min. The flow rate was 1 mL/min, and the detector was set
at 276 nm.
Isolation and Characterization of BexG2 Product (16). A
large-scale BexG2 reaction (1 mL) containing 2-thio-D-glucose (3,
1
2
0 mM), ATP (12 mM), UDP-D-glucose (15, 12 mM), MgCl (3
mM), hexokinase (20 µg), and BexG2 (30 µM) in 100 mM
Tris·HCl buffer, pH 8.0, was carried out. The reaction mixture was
incubated at 30 °C for 6.5 h and filtered through an Amicon
ultrafiltration unit equipped with a YM-10 membrane to remove
the proteins. HPLC analysis and purification were performed using
a Dionex CarboPac PA1 semipreparative column (9 × 250 mm).
The sample (330 µL × 3) was eluted with 0.1 M NH
4
OAc at 5
mL/min. The resulting flow through was split to a collection fraction
4.85 mL/min) and a detection fraction (0.15 mL/min) by a splitter.
(
The elution was monitored by a Corona CAD. The retention time
2
in the presence of Dowex 50W X8 for 20 h under N . The resulting
of 16 was 10.4 min under the HPLC conditions. The fraction
solution was filtered and lyophilized (260 mg). A part of the residue
100 mg) was used for further purification by silica gel column
1
containing 16 was collected and lyophilized. H NMR (500 MHz,
(
D
2
O) δ 5.11 (d, J ) 3.5 Hz, 1H, 1-H), 5.06 (d, J ) 3.8 Hz, 1H,
′-H), 3.96 (m, 2H, 61,2-H), 3.93 (m, 1H, 5′-H), 3.85 (m, 1H, 5-H),
.76 (dd, J ) 12.5, 2.2 Hz, 1H, 6′ -H), 3.68 (m, 1H, 3′-H), 3.66
-H), 3.53 (dd, J ) 9.9, 3.8 Hz, 1H,
′-H), 3.44 (dd, J ) 9.9, 9.2 Hz, 1H, 4-H), 3.34 (dd, J ) 10.1, 9.2
chromatography (water-acetonitrile, 1:50). The product 3 was
obtained as a white powder (30 mg, 0.15 mmol, 32%). In solution,
3
1
3
1
1
exists as a mixture of R and ꢀ isomers (R/ꢀ ) 9:10, under the H
(
m, 1H, 3-H), 3.65 (m, 1H, 6′
2
1
NMR conditions). H NMR (500 MHz, D
Hz, 1H, 1 -H), 4.55 (d, J ) 8.6 Hz, 1H, 1 -H), 3.75 (dd, J ) 12.3,
.3 Hz, 1H, 6ꢀ1-H), 3.74 (m, 1H, 5 -H), 3.69 (dd, J ) 12.3, 2.4
2
O) δ 5.13 (d, J ) 3.3
2
R
ꢀ
13
Hz, 1H, 4′-H), 2.90 (dd, J ) 10.8, 3.5 Hz, 1H, 2-H); C NMR
125 MHz, D O) δ 94.8 (1-C), 93.4 (1′-C), 73.8 (3-C), 72.9 (5′-
C), 72.7 (3′-C), 71.9 (5-C), 71.1 (2′-C), 70.4 (4-C), 69.8 (4′-C),
2
R
(
2
Hz, 1H, 6R1-H), 3.62 (dd, J ) 12.3, 5.1 Hz, 1H, 6R2-H), 3.58 (dd,
J ) 12.3, 5.9 Hz, 1H, 6ꢀ2-H), 3.48 (dd, J ) 10.7, 9.0 Hz, 1H,
-
6
3.8 (6-C), 60.8 (6′-C), 44.5 (2-C); HRMS (ESI ) calcd for
-
3
3
3
R
-H), 3.34 (ddd, J ) 9.7, 5.9, 2.3 Hz, 1H, 5ꢀ2-H), 3.27 (m, 1H,
12 22
C H O
13SP [M - H]- 437.0524, found 437.0520.
ꢀ
-H), 3.26 (m, 1H, 4
R
-H), 3.23 (m, 1H, 4
ꢀ
-H), 2.75 (dd, J ) 10.7,
-H); 13C NMR
-C), 76.7 (5
-C), 61.6 (6 -C),
.3 Hz, 1H, 2
R
-H), 2.57 (dd, J ) 10.2, 8.6 Hz, 1H, 2
ꢀ
Results and Discussion
(
125 MHz, D
C), 74.6 (3 -C), 72.8 (5
1.4 (6 -C), 48.5 (2 -C), 45.8 (2
2
O) δ 98.2 (1
ꢀ
-C), 94.5 (1
R
-C), 77.2 (3
ꢀ
ꢀ
-
Production, Isolation, and Identification of BE-7585A. To
determine that the A. orientalis subsp. Vinearia BA-07585 strain
can produce BE-7585A (1) in our hand and to verify the
assigned structure, the bacterial strain was grown in ISP-2
medium. The major product, which is red, was purified and
subjected to spectroscopic analyses. The HRMS results (HRMS
R
R
-C), 71.5 (4
R
-C), 71.2 (4
ꢀ
ꢀ
+
6
R
ꢀ
R
-C); HRMS (ESI ) calcd for
+
+
-
C H O NaS [M + Na] 219.0298, found 219.0303. (ESI ) calcd
6 12 5
6 11 5
for C H O S
[M - H]- 195.0333, found 195.0330.
-
BexG2 in Vitro Activity Assay. As depicted in Scheme 2, a
typical BexG2 assay mixture (0.1 mL) contained 2-thio-D-glucose
(
3, 1 mM), ATP (1 mM), UDP-D-glucose (15, 1 mM), MgCl
2
(10
+
+
(
8
8
ESI ) calcd for C37
H
46
O
20NaS [M + Na] 865.2201, found
mM), hexokinase (5 µg), and BexG2 (16 µM) in 50 mM Tris ·HCl
buffer, pH 8.0. The substrate for BexG2, 2-thio-D-glucose 6-phos-
phate (14), was produced in situ from 3 and ATP by hexokinase.
The reaction mixture was incubated at 30 °C for 1.5 h and filtered
through an Amicon ultrafiltration unit equipped with a YM-10
membrane to remove the proteins. HPLC analysis was performed
using a Dionex CarboPac PA1 analytical column (4 × 250 mm).
The sample was eluted with a gradient of water (solvent A) and 1
-
-
45
65.2208; HRMS (ESI ) calcd for C37H O20S [M - H]
41.2225, found 841.2222) are consistent with the reported
structure. Further verification of the structure of 1 relied on a
series of NMR experiments (see Figures S1-6 in Supporting
1
13
35
13
Information for H and C NMR spectra). The C chemical
shift of C-2′ at δ 52.4 is further upfield than that typically
expected for an O-methine carbon and thus argues for the
J. AM. CHEM. SOC. 9 VOL. 132, NO. 21, 2010 7409

A R T I C L E S
Sasaki et al.
into the benz[a]anthracene backbone (19) in a manner in which
the head (C-20) and the tail (C-2) of the decaketide chain
coincide with C-13 and C-2, respectively, in the cyclic product
(
18 f 19). A decarboxylation of the last acetate unit (at C-2 to
release C-1) is a necessary step in this pathway to account for
the incorporation pattern. While such a mode of skeletal
assembly involving simple folding and condensation is found
for most augucycline-type natural products studied thus far, an
alternative pathway has been found in the biosynthesis of
PD116198 (32, see Scheme 4) from Streptomyces phaeochro-
4
2
mogenes WP 3688. In this case, chain elongation and ring
condensation lead to a linearly fused tetracyclic anthracyclinone
intermediate (20). Subsequent oxidative bond cleavage between
C-10a and C-11 followed by rearrangement and recylcization
are proposed to give the angular angucyclinone core (20 f 21,
Scheme 3). Since BE-7585A displays the same acetate incor-
poration pattern as PD116198 (32), an analogous ring recon-
struction via an enzyme-catalyzed Baeyer-Villiger oxidation
may also be operative in the biosynthesis of 1. Similar oxidative
C-C bond cleavages of polyketide-derived tetracyclic substrates
have also been proposed for the biosynthesis of gilvocarcin (30)
Figure 2. Selected HMBC results for BE-7585A (1) and selected NOESY
results for 16.
presence of a thiol substituent at this position rather than a
hydroxyl group. The connectivity of the three sugar moieties
in the structure is established by HMBC analysis. The results
3
5
(
listed in Table S1) clearly reveal the presence of a 1′,1′′-O-
glycosyl bond between the 2-thioglucose and the glucose
moieties (see Figure 2). The JH-H values (3.1-3.5 Hz) of the
3
and jadomycin (31) based on gene disruption and gene comple-
anomeric protons (H-1′, H-1′′, and H-1′′′) and the NOESY
experiment established an R,R-linkage between the disaccharide
and an anomeric R-configuration of the rhodinose moiety (2)
to C-12b of the aglycone can also be deduced. The observed
HMBC correlation between H-2′ and C-5 implicates a thioether
linkage between the 2-thiosugar and the aglycone. These results
clearly distinguish the structure of BE-7585A (1) from its
constitutional isomer, rhodonocardin A (5), and thus exclude
the possibility that 1 is identical to rhodonocardin A.
43,44
mentation experiments.
Enzymes responsible for this type
of reactions are flavin-dependent monooxygenases. Notably, a
flavin monooxygenase (MtmOIV) catalyzing the Baeyer-Villiger
ring-opening in the biosynthesis of mithramycin (33) was
45,46
recently characterized and its crystal structure determined.
Identification of the Gene Cluster. As mentioned above,
47
urdamycin A (17) is an angucycline class antibiotic having a
similar structure as BE-7585A (1). It is composed of a
benz[a]anthraquinone core, two L-rhodinose (2), and two
D-olivose moieties, with one of the rhodinosyl groups attached
at the C-12b position, as seen in the structure of 1. The gene
cluster of 17 has been identified and sequenced, and the
1
3
C-Labeling Experiments. The angucycline core structures
are generally derived from the assembly of 10 acetate units
36
catalyzed by type II polyketide synthases (PKSs). To confirm
the biosynthetic origin of the ring carbons of BE-7585A (1),
48
biosynthetic pathway of 17 has been studied. In view of the
1
3
[
1- C]acetate was added into a growing A. orientalis culture.
close resemblance between 17 and 1, homologous genes
encoding the type II polyketide synthase (PKS), the aglycone
tailoring enzymes, TDP-rhodinose biosynthetic enzymes, and
a rhodinosyltransferase found in the urdamycin gene cluster are
expected to exist in the gene cluster of 1 as well. To locate the
BE-7585A biosynthetic gene cluster, a cosmid library was
constructed from the genomic DNA of A. orientalis. This library
was screened using PCR probes designed based on multiple
sequence alignment of several KSRs, hexose 2,3-dehydratases
1
3
13
The C-labeled product was purified and analyzed by C NMR
spectroscopy. Enhanced signals for C-1, C-3, C-4a, C-6, C-7,
C-8, C-10, C-11a, and C-12a of the ring structure of 1 are
observed, indicating these carbons are derived from C-1 of
acetate (Scheme 3, and see Figure S7). To determine the
orientation of these acetate units during assembly, [1,2-
C ]acetate was also utilized in the feeding experiment. The
2
C- C spin couplings were observed for C-1/C-2, C-3/C-13,
C-4a/C-12b, C-5/C-6, C-6a/C-7, C-7a/C-8, C-9/C-10, C-11/C-
3
5
1
1
3
3
13
3
5
1
1a, and C-12/C-12a (see Figure S8), indicating that these
(
40) (a) Sato, Y.; Geckle, M.; Gould, S. J. Tetrahedron Lett. 1985, 26,
pairs of carbons are derived from intact acetate units. This
assignment was confirmed by the INADEQUATE spectrum (see
4
023–4026. (b) Sato, Y.; Gould, S. J. J. Am. Chem. Soc. 1986, 108,
625–4631. (c) Seaton, P. J.; Gould, S. J. J. Am. Chem. Soc. 1987,
4
3
5
Figure S9). Thus, the position and orientation of acetate units
in the angucycline core structure of 1 were unambiguously
determined.
109, 5282–5284.
(
41) (a) Takahashi, K.; Tomita, F. J. Antibiot. 1983, 36, 1531–1535. (b)
Carter, G. T.; Fantini, A. A.; James, J. C.; Borders, D. B.; White,
R. J. J. Antibiot. 1985, 38, 242–248.
Interestingly, the acetate incorporation pattern observed here
is unusual for angucycline class natural products (see Scheme
(42) Gould, S. J.; Halley, K. A. J. Am. Chem. Soc. 1991, 113, 5092–5093.
(43) Liu, T.; Fischer, C.; Beninga, C.; Rohr, J. J. Am. Chem. Soc. 2004,
126, 12262–12263.
3
7
3
). As exemplified by the biosynthesis of urdamycin A (17),
(
44) Rix, U.; Wang, C.; Chen, Y.; Lipata, F. M.; Rix, L. L. R.; Greenwell,
L. M.; Vining, L. C.; Yang, K.; Rohr, J. ChemBioChem 2005, 6, 838–
845.
38 39 40
1
vineomycin A , PD116740, kinamycin D, and gilvocarcin
V (30), in the typical ring closure reaction, the linear
decaketide (18) formed through the action of PKS is cyclized
4
1
(
45) Gibson, M.; Nur-e-alam, M.; Lipata, F.; Oliveira, M. A.; Rohr, J. J. Am.
Chem. Soc. 2005, 127, 17594–17595.
(
46) Beam, M. P.; Bosserman, M. A.; Noinaj, N.; Wehenkel, M.; Rohr, J.
Biochemistry 2009, 48, 4476–4487.
(
(
35) See Supporting Information.
36) Hertweck, C.; Luzhetskyy, A.; Rebets, Y.; Bechthold, A. Nat. Prod.
Rep. 2007, 24, 162–190.
(47) Drautz, H.; Z a¨ hner, H.; Rohr, J.; Zeeck, A. J. Antibiot. 1986, 12, 1657–
1669.
(
(
(
37) Rohr, J. J. Antibiot. 1989, 42, 1151–1157.
38) Tanaka, H.; Omura, S. J. Antibiot. 1982, 35, 602–608.
39) Gould, S. J.; Cheng, X. C.; Halley, K. A. J. Am. Chem. Soc. 1992,
(48) (a) Decker, H.; Haag, S. J. Bacteriol. 1995, 177, 6126–6136. (b) Faust,
B.; Hoffmeister, D.; Weitnauer, G.; Westrich, L.; Haag, S.; Schneider,
P.; Decker, H.; K u¨ nzel, E.; Rohr, J.; Bechthold, A. Microbiology 2000,
146, 147–154.
1
14, 10066–10068.
7
410 J. AM. CHEM. SOC. 9 VOL. 132, NO. 21, 2010

Biosynthesis of BE-7585A
A R T I C L E S
Scheme 3
and hexose 3-dehydrases found in the gene clusters of 17 and
several other related compounds. Five cosmids were found to
harbor these genes. Sequencing of these cosmids led to a
contiguous DNA sequence of 48.4 kb, from which 42-open
reading frames (ORFs) (orf1-42) were identified (Figure 3).
Among them, orf9-36 are believed to be involved in BE-7585A
biosynthesis based on BLAST search (Table 2). As predicted,
the cluster includes type II PKSs, post-PKS aglycone tailoring
genes, and the deoxysugar biosynthetic genes. The remaining
ORFs (orf1-8 and orf37-42) are likely boundary ORFs, since
they appear to be the biosynthetic genes for primary metabolites
R ꢀ
minimal PKS, KS , ꢀ-ketoacyl synthase ꢀ subunit (KS ), and
acyl carrier protein (ACP), respectively. The minimal PKS
(BexA, BexB, and BexC) assembles 10 malonyl-CoAs from
10 acetate units into a linear decaketide chain (18). As shown
in Scheme 4, to accommodate the unusual labeling pattern
observed in the 13C-labeling experiments (see Scheme 3), 18
should be cyclized to a linear structure 23 rather than an angular
structure (i.e., 19). On the basis of the established biosynthesis
of other angucycline type compounds, a ketoreductase homo-
logue, BexD, and two cyclase homologues, BexL and BexF,
are predicted to act together with PKS to construct the ring
3
5
36
(
See Table S2). Overall, the BE-7585A biosynthetic gene
structure. Specifically, the reduction of the 9-keto group by
cluster (bex cluster) spans a region of 28.9 kb and contains 28
ORFs. The nucleotide sequences reported herein have been
deposited into the GenBank database under the accession
number HM055942.
Functional Predication of Genes Involved in Angucycline
Core Formation. The deduced gene products of bexA, bexB,
and bexC display high sequence similarities to the type II
BexD likely initiates the ring formation reaction catalyzed by
BexL. Subsequent decarboxylation and cyclization by BexF
yields 23 as a key intermediate.
The resulting ring structure 23 would then be tailored by a
set of oxygenases to the angular angucyclinone core (29). Recent
studies indicated that multioxygenase complexes are required to
perform the oxidative C-C bond cleavage and ring rearrangement
J. AM. CHEM. SOC. 9 VOL. 132, NO. 21, 2010 7411

A R T I C L E S
Sasaki et al.
Scheme 4
4
4,49
in both gilvocarcin (30) and jadomycin (31) biosynthesis.
Since
Other genes likely involved in aglycone biosynthesis are
bexK, bexN, and bexW. BexK is a cytochrome P450 hydroxylase
homologue, which may be responsible for hydroxylation at C-2
of 28 to produce 29. This assignment is supported by an early
observation that a cytochrome P450 inhibitor blocked C-2
hydroxylation in PD116198 (32) biosynthesis, implicating C-2
a similar oxidative ring-opening process (23 f 29) likely occurs
in the formation of BE-7585A, a complex of oxygenases may also
be involved. A few possible candidates include a flavin-dependent
oxygenase homologue, BexE, a bifunctional oxygenase-reductase
homologue, BexM, and the anthrone monooxygenase homologue,
BexI, which displays sequence similarity to GilOII and JadG from
51
hydroxylation in both pathways as a P450-dependent reaction.
4
4,50
the gilvocarcin and jadomycin pathways, respectively.
Although
BexN is an acetyl-CoA carboxylase homologue, which may be
involved in supplying malonyl-CoA in PKS-catalyzed reactions.
Precedence is found in the jadomycin pathway where a BexN
it is difficult to define the cascade of the oxidation events solely
based on sequence analysis, the identification of genes encoding
these oxygenases (BexE, BexI, and BexM) in the bex cluster
provides important leads for future biochemical experiments to
address these tailoring oxidation reactions in the biosynthesis of
BE-7585A (1).
52
homologue, JadN, is believed to play a similar role. BexW is
a NADPH-dependent FMN reductase homologue, which may
serve as the reductase for the flavin-dependent oxygenases
involved in the aglycone biosynthesis.
(
(
49) Fischer, C.; Lipata, F.; Rohr, J. J. Am. Chem. Soc. 2003, 125, 7818–
819.
50) Kharel, M. K.; Zhu, L.; Liu, T.; Rohr, J. J. Am. Chem. Soc. 2007,
29, 3780–3781.
7
(51) Gould, S.; Cheng, X. C. J. Org. Chem. 1994, 59, 400–405.
(52) Wang, L.; McVey, J.; Vining, L. C. Microbiology 2001, 147, 1535–
1545.
1
7
412 J. AM. CHEM. SOC. 9 VOL. 132, NO. 21, 2010

Biosynthesis of BE-7585A
A R T I C L E S
Figure 3. Organization of the BE-7585A biosynthetic gene cluster (bex cluster). The gene cluster spans 28.9 kbp and contains 28 open reading frames.
Table 2. Proposed Functions of ORFs in the BE-7585A Biosynthetic Cluster (bex Cluster)
gene
proposed function
protein homologue and origin
identity/similarity (%) protein accession number
bexR1 regulator
repressor-response regulator [Streptomyces sp. AM-7161]
putative oxygenase (Aur1A) [Streptomyces aureofaciens]
putative cyclase (UrdF) [Streptomyces fradiae]
putative ketoacyl synthase (UrdA) [Streptomyces fradiae]
keto-acyl synthase beta [Streptomyces antibioticus]
putative acyl carrier protein (SimA3) [Streptomyces antibioticus]
ketoreductase [Streptomyces antibioticus]
putative aromatase [Streptomyces sp. SCC 2136]
oxygenase-reductase (UrdM) [Streptomyces fradiae]
Transporter (UrdJ2) [Streptomyces fradiae]
56/68
75/83
78/85
85/92
74/83
60/74
81/89
70/78
62/71
47/61
43/54
48/65
BAC79018
AAX57188
CAA60568
CAA60569
CAG14966
AAK06786
CAG14968
CAH10113
AAF00206
AAF00207
ZP_05020741
ZP_04471118
bexE
bexF
bexA
bexB
bexC
bexD
bexL
bexM
oxygenase
cyclase
ketosynthase R subunit
ketosynthase ꢀ subunit
acyl carrier protein
ketoreductase
cyclase
oxygenase-reductase
bexJ1 transporter
bexN
bexH
carboxyl transferase
decarboxylase (JadN) [Streptomyces sViceus ATCC 29083]
sugar epimerase/reductase predicted nucleoside-diphosphate sugar epimerase [Streptosporangium
roseum DSM 43021]
bexI
bexK
oxygenase
hydroxylase
putative anthrone monooxygenase (GilOII) [Streptomyces griseoflaVus]
cytochrome P-450-like enzyme [Saccharopolyspora erythraea NRRL
27/38
41/54
AAP69583
YP_001107923
2
338]
bexO
bexP
ferredoxin
methyltransferase
ferredoxin [Streptomyces coelicolor A3(2)]
putative methyltransferase [Saccharopolyspora erythraea
NRRL 2338 ]
40/59
37/52
NP_625075
YP_001105373
bexG1 glycosyltransferase
glycosyl transferase (UrdGT1a) [Streptomyces fradiae]
sugar 3-ketoreductase (KijD10) [Actinomadura kijaniata]
putative dNDP-glucose 4,6-dehydratase [Streptomyces aureofaciens]
NDP-hexose 3,4-dehydratase homologue [Streptomyces cyanogenus]
putative TDP-glucose synthase [Streptomyces nogalater]
NDP-hexose 2,3-dehydratase [Frankia sp. CcI3]
TetR family transcriptional regulator [Saccharopolyspora erythraea
NRRL 2338]
49/62
52/65
72/80
78/86
69/82
58/72
50/61
AAF00214
ACB46498
ACK77743
AAD13547
AAF01820
YP_483211
YP_001103502
bexQ
bexS
bexT
bexU
bexV
sugar 3-ketoreductase
sugar 4,6-dehydratase
sugar 3-dehydrase
TDP-glucose synthase
sugar 2,3-dehydratase
bexR2 regulator
bexW
bexJ2 transporter
bexX
reductase
putative reductase [Streptomyces griseoflaVus]
EmrB/QacA family drug resistance transporter [Frankia sp. EAN1pec]
Thiazole biosynthesis protein (ThiG) [Stigmatella aurantiaca DW4/3-1]
major facilitator superfamily transporter [Mycobacterium Vanbaalenii
PYR-1]
67/74
36/53
58/75
36/50
AAP69587
YP_001507963
ZP_01459245
YP_956695
thiosugar synthase
bexJ3 transporter
bexG2 glycosyltransferase
R,R-trehalose-phosphate synthase [Methanothermobacter
thermautotrophicus str. Delta H]
30/47
NP_276863
2
0b,d
Functional Predication of Genes Involved in Rhodinose
Formation and Rhodinosyltransfer. Rhodinose and rhodinose-
derivatives are found in several natural products whose bio-
deoxyhexoses, to TDP-L-rhodinose (40).
The pathway is
initiated by the activation of 34 to TDP-glucose (35), catalyzed
by a TDP-glucose synthase homologue, BexU. The second step
is the conversion of 35 to 36 catalyzed by a TDP-glucose 4,6-
dehydratase homologue, BexS. Subsequent C-2 deoxygenation
(36 f 37) is catalyzed by a 2,3-dehydratase homologue, BexV,
and a 3-ketoreductase homologue, BexQ. The resulting product
37 then undergoes C-3 deoxygenation to 38 catalyzed by a
[2Fe-2S]-containing and PMP-dependent BexT, which displays
good sequence homology to hexose 3-dehydrase enzymes (78%
identity to LanQ from S. cyanogenus; 73% identity to SpnQ
27,28,53-55
synthetic gene clusters have been isolated and sequenced.
Sequence comparison of the putative sugar biosynthetic genes
in these clusters and those in the bex cluster allowed the
identification of the rhodinose biosynthetic genes. Scheme 5
shows the correlation of their proposed functions and the
chemical transformation steps required to convert glucose
1
-phosphate (34), which is the biosynthetic precursor for most
2
6,56
(
(
53) R a¨ ty, K.; Kunnari, T.; Hakala, J.; M a¨ nts a¨ l a¨ , P.; Ylihonko, K. Mol.
Gen. Genet. 2000, 264, 164–172.
from Saccharopolyspora spinosa).
While this reaction
typically requires an iron-sulfur containing flavoprotein re-
ductase for coenzyme regeneration, no gene encoding such a
54) Ichinose, K.; Bedford, D. J.; Tornus, D.; Bechthold, A.; Bibb, M. J.;
Revill, W. P.; Floss, H. G.; Hopwood, D. A. Chem. Biol. 1998, 5,
57
6
47–659.
specific reductase can be located in the bex cluster. It is possible
(
55) Olano, C.; G o´ mez, C.; P e´ rez, M.; Palomino, M.; Pineda-Lucena, A.;
Carbajo, R. J.; Bra n˜ a, A. F.; M e´ ndez, C.; Salas, J. A. Chem. Biol.
2
009, 16, 1031–1044.
(56) Thorson, J. S.; Liu, H.-w. J. Am. Chem. Soc. 1993, 115, 7539–7540.
J. AM. CHEM. SOC. 9 VOL. 132, NO. 21, 2010 7413

A R T I C L E S
Sasaki et al.
Scheme 5
Scheme 6
that a general cellular reductase or BexO, which is a ferredoxin
homologue, assumes this role.
The bex cluster also contains the gene bexH that is believed
to encode a sugar epimerase. Thus, a 5-epimerization reaction
to the C-12b position of the angucycline core (29) to generate
41. Since the UrdGT1a reaction proceeds with inversion of
stereochemistry at the anomeric center of rhodinose and both
BexG1 and UrdGT1a are members of the GT-1 glycosyltrans-
ferase family of enzymes, BexG1 is expected to be an inverting
enzyme as well. This implies that the TDP leaving group of
the sugar donor should be ꢀ-oriented as shown in 40, because
the glycosidic linkage of the rhodinose moiety in 41 is in the
R-configuration. An epimerization at C-5 of 38 is thus necessary
to fix the anomeric configuration as ꢀ in 39 and 40.
Biosynthesis of the Thiosugar Moiety. Among all ORFs in
the bex cluster, the bexX gene, which displays good sequence
similarity to thiazole synthases, ThiG (58% identity to ThiG
from Stigmatella aurantiaca DW4/3-1; 38% identity to ThiG
from Bacillus subtilis subsp. subtilis str 168) is clearly important
for the biosynthesis of the thiosugar (3). ThiG is a key enzyme
involved in the formation of thiazole of thiamine. Studies of
thiamine biosynthesis in B. subtilis showed that the ThiG
reaction is initiated by the Schiff base formation between one
of lysine residues (Lys96) of ThiG and the 2-keto group of its
substrate, 1-deoxy-D-xylulose-5-phosphate (DXP, 42). As il-
lustrated in Scheme 6, the sulfur atom is transferred from the
thiocarboxylate (43) at the C-terminus of a sulfur carrier protein,
2
6,58
61
(
38 f 39) followed by 4-ketoreduction (39 f 40) is proposed
to complete the biosynthesis of TDP-L-rhodinose (40). However,
no hexose 4-ketoreductase gene can be located in the bex cluster.
Whether the 4-ketoreduction of 39 is mediated by a nonspecific
reductase in the cellular pool or BexH functions as a bifunctional
5
9
epimerase-ketoreductase will be determined in future bio-
chemical studies. Further support for the assignment of 40 as
an L-sugar comes from another gene product in the bex cluster,
BexG1. This glycosyltransferase homologue demonstrates high
sequence similarity to the TDP-L-rhodinosyltransferase, UrdGT1a,
from the urdamycin pathway having 49% identity and 62%
6
0
similarity. Therefore, BexG1 is predicted to have a similar
function catalyzing the transfer of the L-rhodinosyl group of 40
(
(
(
57) (a) Hallis, T. M.; Liu, H.-w. Acc. Chem. Res. 1999, 32, 579–588. (b)
He, X.; Agnihotri, G.; Liu, H.-w. Chem. ReV. 2000, 100, 4615–4661.
58) Hong, L.; Zhao, Z.; Melanµon, C. E., III.; Zhang, H.; Liu, H.-w. J. Am.
Chem. Soc. 2008, 130, 4954–4967.
59) (a) Chang, S.; Duerr, B.; Serif, G. J. Biol. Chem. 1988, 263, 1693–
1
1
697. (b) Alam, J.; Beyer, N.; Liu, H.-w. Biochemistry 2004, 43,
6450–16460.
(
60) Trefzer, A.; Hoffmeister, D.; K u¨ nzel, E.; Stockert, S.; Weitnauer, G.;
Westrich, L.; Rix, U.; Fuchser, J.; Bindseil, K. U.; Rohr, J.; Bechthold,
A. Chem. Biol. 2000, 7, 133–142.
(61) Schuman, B.; Alfaro, J. A.; Evans, S. V. Top. Curr. Chem. 2007, 272,
217–257.
7
414 J. AM. CHEM. SOC. 9 VOL. 132, NO. 21, 2010

Biosynthesis of BE-7585A
A R T I C L E S
Scheme 7
6
2,63
ThiS, and incorporated into the ThiG-DXP adduct (44).
trehalose. The latter is commonly formed by linking the glucose
moiety of UDP-glucose (15) to glucose 6-phosphate (45) and
the reaction is catalyzed by trehalose 6-phosphate synthases
Because of the presence of the bexX gene in the cluster, an
analogous mechanism of sulfur incorporation can be proposed
for the biosynthesis of the 2-thioglucose (Scheme 7). Accord-
ingly, the putative substrate, glucose 6-phosphate (45), may form
an imine adduct (46) with the active site lysine of BexX at C-1
position. Deprotonation of C-2 of 46 followed by tautomeriza-
tion via an enamine 47 generates a 2-keto intermediate 48, which
can then react with a nucleophilic sulfur group to incorporate a
sulfur atom at C-2 in 49. The reactive sulfur donor is likely a
ThiS-thiocarboxylate or a protein persulfide equivalent. Dehy-
dration of 49 to 50 followed by C-1 deprotonation gives an
enamine intermediate 51, which can tautomerize to C-1 imine
6
5
(TPSs). Since the bexG2-encoded protein exhibits sequence
similarity to TPSs (30% identity and 47% similarity to TPS
from Methanothermobacter thermautotrophicus str. Delta H),
BexG2 is likely the 2-thiotrehalose 6-phosphate synthase
catalyzing the coupling of 14 and 15 to form 16, whose
6-phosphate group is removed at a later stage. In archaea,
bacteria, fungi, plants, and animals, the dephosphorylation of
trehalose 6-phosphate to trehalose is catalyzed by trehalose
6
5
6-phosphate phosphatase (TPP). In fact, the TPS- and TPP-
encoding genes are clustered in Streptomyces aVermitilis and
6
6,67
5
2. Upon hydrolysis, the BexX-bound product 52 is released
Saccharopolyspora erythraea genomes.
Although no TPP
to give 2-thioglucose 6-phosphate (53/14).
homologous gene is found in the bex cluster or in the boundary
region, the phosphate group of 16 may be hydrolyzed by an
endogenous phosphatase encoded elsewhere in the genome. The
hydrolysis may occur on the disaccharide 16 or after the
disaccharide has been attached to the aglycone. Interestingly, a
new trehalose biosynthetic route (catalyzed by TreTs), which
utilizes UDP-glucose (15) and glucose instead of glucose
The sulfur atom of ThiS-thiocarboxylate has been shown to
be derived from cysteine mediated by a cysteine desulfurase.
In B. subtilis (and E. coli), this sulfur loading reaction also
6
2
6
4
requires ThiF, which plays a role in ThiS activation. Unfor-
tunately, neither ThiS homologue nor a cysteine desulfurase
homologue is found in the bex cluster. However, since the
substrate and the function of ThiG and BexX are analogous, it
is possible that the endogenous ThiS used in thiamin biosyn-
thesis may be recruited to serve as the sulfur donor for thiosugar
biosynthesis in A. orientalis. Further investigation of the sulfur
delivery process is in progress.
6
8
6
-phosphate (45), was recently found. However, the poor
sequence alignment results between BexG2 and these trehalose
glycosyltransferring synthases (TreTs) renders this route less
likely for the BexG2 reaction.
Pathway of the Disaccharide Formation and Its Transfer
to the Aglycone. Once formed, the BexX product, 2-thioglucose
-phosphate (14), is expected to accept a glucose moiety from
UDP-glucose (15) to form 2-thiotrehalose 6-phosphate (16). This
disaccharide moiety of BE-7585A (1) is structurally related to
(
65) Avonce, N.; Mendoza-Vargas, A.; Morett, E.; Iturriaga, G. BMC EVol.
Biol. 2006, 6, 109.
6
(66) Ikeda, H.; Ishikawa, J.; Hanamoto, A.; Shinose, M.; Kikuchi, H.; Shiba,
T.; Sakaki, Y.; Hattori, M.; Omura, S. Nat. Biotechnol. 2003, 21, 526–
5
31.
(
67) Oliynyk, M.; Samborskyy, M.; Lester, J. B.; Mironenko, T.; Scott,
N.; Dickens, S.; Haydock, S. F.; Leadlay, P. F. Nat. Biotechnol. 2007,
25, 447–453.
(
(
62) Begley, T. P. Nat. Prod. Rep. 2006, 23, 15–25.
63) Settembre, E. C.; Dorrestein, P. C.; Zhai, H.; Chatterjee, A.; McLaf-
ferty, F. W.; Begley, T. P.; Ealick, S. E. Biochemistry 2004, 43, 11647–
(68) (a) Qu, Q.; Lee, S.-J.; Boos, W. J. Biol. Chem. 2004, 279, 47890–
47897. (b) Ryu, S.-I.; Park, C.-S.; Cha, J.; Woo, E.-J.; Lee, S.-B.
Biochem. Biophys. Res. Commun. 2005, 329, 429–436. (c) Kouril, T.;
Zaparty, M.; Marrero, J.; Brinkmann, H.; Siebers, B. Arch. Microbiol.
2008, 190, 355–369. (d) Nobre, A.; Alarico, S.; Fernandes, C.;
Empadinhas, N.; da Costa, M. S. J. Bacteriol. 2008, 190, 7939–7946.
1
1657.
(
64) (a) Xi, J.; Ge, Y.; Kinsland, C.; McLafferty, F. W.; Begley, T. P.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 8513–8518. (b) Lehmann, C.;
Begley, T. P.; Ealick, S. E. Biochemistry 2006, 45, 11–19.
J. AM. CHEM. SOC. 9 VOL. 132, NO. 21, 2010 7415

A R T I C L E S
Sasaki et al.
Scheme 8
Figure 4. BexG2 activity and substrate specificity assay. (A) In situ
production of 2-thioglucose 6-phosphate (14) from 2-thioglucose (3) using
hexokinase was monitored by the consumption of ATP (21.4 min) and the
production of ADP (16.5 min) at 276 nm. The conversion of 14 to 16
catalyzed by BexG2 was detected by the consumption of UDP-glucose (15)
(
6.1 min) and the production of UDP (10.5 min) at 276 nm. (a) Control
reaction, no enzymes; (b) control reaction, no BexG2; (c) the complete
reaction containing 3 (1 mM), ATP (1 mM), 15 (1 mM), MgCl (10 mM),
hexokinase (5 µg), and BexG2 (16 µM) in 50 mM Tris ·HCl buffer, pH 8.0
0.1 mL); (d) control reaction to investigate the substrate specificity, no
ATP and hexokinase. (B) Isolation of BexG2 product 16. The large-scale
reaction contained 3 (10 mM), ATP (12 mM), 15 (12 mM), MgCl (3 mM),
2
(
2
hexokinase (20 µg), and BexG2 (30 µM) in 100 mM Tris cdtHCl buffer,
pH 8.0 (1 mL). The resulting product 16 (10.4 min, monitored by the Corona
CAD) was collected and characterized.
and 15 and the production of ADP and UDP (Figure 4A, trace
c). This observation indicated that the tandem reactions carried
out by hexokinase and BexG2 were successful. In the absence
of BexG2, only consumption of ATP and the production of ADP
were observed (Figure 4A, trace b). Therefore, the conversion
of 15 to UDP must be BexG2-dependent. When both enzymes
were omitted in a control, no reaction occurred (Figure 4A, trace
a). To confirm the identity of the reaction product, a Corona
CAD was used to detect product formation. As shown in Figure
How the thiodisaccharide (16 or its hydrolyzed product
without 6-phosphate group) is attached to C-5 of the benz[a]an-
thraquinone core is not clear. It is conceivable that the thioether
bond formation may simply be a result of a nonenzymatic attack
by the nucleophilic 2-thiol group of the disaccharide at the C-5
position of benz[a]anthraquinone (29 or 41). The C-5 site is
expected to be electrophilic and thus can function as a Michael
acceptor. Subsequent deprotonation at C-5 leads to a hydro-
quinone intermediate (54), which can be readily oxidized to the
quinone form (55) by molecular oxygen (see Scheme 8). A
similar path has been demonstrated in urdamycin E (56)
4B, a new peak was observed in the hexokinase-BexG2 reaction.
The corresponding fraction was collected, and the isolated
product was subjected to NMR and MS analyses. All data are
consistent with the assigned structure, 16, which is linked by
an R,R-1′,1′′-glycosidic bond (see Figure 2).
We then tested if 2-thioglucose (3) can be processed by
BexG2. The reaction conditions were the same as described
above except for the omission of hexokinase and ATP. HPLC
analysis of the reaction showed that no UDP was produced
6
9
biosynthesis.
Regulatory and Resistant Genes. The remaining genes in the
BE-7585A gene cluster are mainly regulatory and resistance
genes. Among them, BexR1 and BexR2 display sequence
similarity to transcriptional regulators. BexJ1, BexJ2, and BexJ3
are transporter homologues and are possibly involved in self-
(Figure 4A, trace d). Thus, it appears that BexG2 cannot process
3
2
as a substrate, and phosphorylation at C-6 position of
-thioglucose is necessary for the BexG2 reaction. This is
consistent with the sequence analysis of BexG2, which exhibits
higher sequence similarity to trehalose phosphate synthases than
70
resistance mechanisms. A methyltransferase homologue, BexP,
may also be involved in the self-resistance mechanism by
catalyzing methylation of rRNA.
68
to trehalose glycosyltransferring synthases (TreTs) as discussed
above.
BexG2 Activity and Substrate Specificity Assay. To verify
the proposed pathway for disaccharide formation, the putative
glycosyltransferase, BexG2, was expressed and purified to near
homogeneity (see Figure 1). The predicted substrate, 2-thio-
glucose 6-phosphate (14), was produced in situ from chemically
synthesized 2-thioglucose (3) using hexokinase (Scheme 2). The
standard reaction mixture contained 14, ATP, UDP-glucose (15),
Conclusions
BE-7585A (1), which is an angucycline-type natural product
produced by A. orientalis subsp. Vinearia BA-07585, contains
a rare 2-thioglucose (3) attached to the polyketide core. In this
work, the BE-7585A biosynthetic cluster (bex cluster) containing
2
8 ORFs was identified by PCR-based cosmid screening of the
MgCl
2
, hexokinase, and BexG2 in Tris ·HCl buffer. HPLC
genomic DNA of the producing strain. The cluster harbors genes
typical for type II polyketide synthesis. Also contained in the
cluster is a thiazole synthase (ThiG) homologue, BexX, which
may catalyze the key reaction producing 2-thioglucose 6-phos-
analysis of the reaction mixture showed the consumption of ATP
(
(
69) Rohr, J. J. Chem. Soc., Chem. Commun. 1989, 8, 492–493.
70) Walsh, C. Nature 2000, 406, 775–781.
7
416 J. AM. CHEM. SOC. 9 VOL. 132, NO. 21, 2010

Biosynthesis of BE-7585A
A R T I C L E S
phate (14) from glucose 6-phoshate (45). On the basis of
sequence analysis, a biosynthetic pathway is proposed for BE-
quantum coherence; INADEQUATE, incredible natural abun-
dance double quantum transfer experiment; IPTG, isopropyl ꢀ-D-
1-thiogalactopyranoside; ISP, International Streptomyces Project;
KSR, ꢀ-ketoacyl synthase R subunit; KSꢀ, ꢀ-ketoacyl synthase
ꢀ subunit; LB, Luria-Bertani; NADPH, nicotinamide adenine
dinucleotide phosphate (reduced form); NCBI, National Center
for Biotechnology Information; NOESY, nuclear Overhauser
enhancement (effect) spectroscopy; NTA, nitrilotriacetic acid;
PCR, polymerase chain reaction; PLP, pyridoxal-5′-phosphate;
SAM, S-adenosyl-L-methionine; SDS-PAGE, sodium dodecyl
sulfate-polyacrylamide gel electrophoresis; TDP, thymidine 5′-
diphosphate; TPP, trehalose 6-phosphate phosphatase; TPS,
trehalose 6-phosphate synthase; TSB, tryptone soya broth; UDP,
uridine 5′-diphosphate.
7
585A including the benz[a]anthraquinone core formation,
rhodinose formation, and 2-thiosugar-containing disaccharide
13
formation. Further isotopic tracer experiments using [1- C] and
13
[
2
1,2- C ]acetate revealed that the construction of the angucy-
cline skeleton involves an unusual oxidative ring-opening and
rearrangement. To obtain support for the proposed biosynthetic
pathways, the disaccharide formation was demonstrated in vitro
using purified enzyme BexG2 and the chemoenzymatically
synthesized 2-thioglucose 6-phosphate. We have also confirmed
that 2-thioglucose 6-phosphate instead of 2-thioglucose is the
substrate for the BexG2 enzyme. Although the direct sulfur
donor has not yet been identified, an endogenous sulfur carrier
protein, such as ThiS used in thiamin biosynthesis, may be
recruited for the thiosugar production. This work provided the
first insights into the biosynthesis of a 2-thiosugar. Studies of
the molecular mechanism of the sulfur incorporation and unusual
angucycline core construction are in progress.
Abbreviations: ACP, acyl carrier protein; BLAST, basic local
alignment search tool; ATP, adenosine 5′-triphosphate; ADP,
adenosine 5′-diphosphate; CAD, charged aerosol detector;
COSY, correlated spectroscopy; DXP, 1-deoxy-D-xylulose-5-
phosphate; ESI, electrospray ionization; FMN, flavin mono-
nucleotide; HMBC, heteronuclear multiple bond correlation;
HPLC, high performance liquid chromatography; HRMS, high
resolution mass spectrometry; HSQC, heteronuclear single
Acknowledgment. We are grateful to Banyu Pharmaceutical
Co. (Tokyo, Japan) for providing the A. orientalis subsp. Vinearia
BA-07585 strain. We also thank David Kim for his assistance with
protein purification. This work was supported in part by the National
Institutes of Health Grants (GM035906).
1
13
Supporting Information Available: H and C NMR spectra;
1
1
H- H COSY, HSQC, HMBC, NOESY spectra of 1; sodium-
13
13
[
2
1- C]acetate, sodium[1,2- C ]acetate feeding experiment;
13
INADEQUATE spectrum of 1 labeled with C. This material
is available free of charge via the Internet at http://pubs.acs.org.
JA1014037
J. AM. CHEM. SOC. 9 VOL. 132, NO. 21, 2010 7417