Involvement of common intermediate 3-hydroxy-L-kynurenine in chromophore biosynthesis of quinomycin family antibiotics

Article abstract of DOI:10.1038/ja.2010.142

Quinomycin antibiotics, represented by echinomycin, are an important class of antitumor antibiotics. We have recently succeeded in identification of biosynthetic gene clusters of echinomycin and SW-163D, and have achieved heterologous production of echinomycin in Escherichia coli. In addition, we have engineered echinomycin non-ribosomal peptide synthetase to generate echinomycin derivatives. However, the biosynthetic pathways of intercalative chromophores quinoxaline-2-carboxylic acid (QXC) and 3-hydroxyquinaldic acid (HQA), which are important for biological activity, were not fully elucidated. Here, we report experiments involving incorporation of a putative advanced precursor, (2S, 3R)-6′- 2 H-3-hydroxy-L-kynurenine, and functional analysis of the enzymes Swb1 and Swb2 responsible for late-stage biosynthesis of HQA. On the basis of these experimental results, we propose biosynthetic pathways for both QXC and HQA through the common intermediate 3-hydroxy-L-kynurenine.

Full text of DOI:10.1038/ja.2010.142

The Journal of Antibiotics (2011) 64, 117–122
2011 Japan Antibiotics Research Association All rights reserved 0021-8820/11 $32.00
www.nature.com/ja
&
ORIGINAL ARTICLE
Involvement of common intermediate
3-hydroxy-L-kynurenine in chromophore biosynthesis
of quinomycin family antibiotics
Yuki Hirose1, Kenji Watanabe2, Atsushi Minami1, Takemichi Nakamura3, Hiroki Oguri1 and Hideaki Oikawa1
Quinomycin antibiotics, represented by echinomycin, are an important class of antitumor antibiotics. We have recently
succeeded in identification of biosynthetic gene clusters of echinomycin and SW-163D, and have achieved heterologous
production of echinomycin in Escherichia coli. In addition, we have engineered echinomycin non-ribosomal peptide synthetase
to generate echinomycin derivatives. However, the biosynthetic pathways of intercalative chromophores quinoxaline-2-carboxylic
acid (QXC) and 3-hydroxyquinaldic acid (HQA), which are important for biological activity, were not fully elucidated. Here, we
report experiments involving incorporation of a putative advanced precursor, (2S, 3R)-[6¢-2H]-3-hydroxy-L-kynurenine, and
functional analysis of the enzymes Swb1 and Swb2 responsible for late-stage biosynthesis of HQA. On the basis of
these experimental results, we propose biosynthetic pathways for both QXC and HQA through the common intermediate
3-hydroxy-L-kynurenine.
The Journal of Antibiotics (2011) 64, 117–122; doi:10.1038/ja.2010.142; published online 24 November 2010
Keywords: biosynthesis; echinomycin; Streptomyces lasaliensis; Streptomyces sp; SW-163D
INTRODUCTION
(HQA, 8)—have been identified (Scheme 1).5 Although biosynthetic
Quinomycin class antibiotics, represented by echinomycin (1)1 and studies of these chromophores have been performed for 111,12 and
SW-163D (2),2–4 are C2-symmetric cyclic depsipeptides with a pair of triostin13 and several gene clusters have been reported,6,9,14,15 little
intercalative chromophores that show potent antibacterial, anticancer experimental data were obtained. Here, we report chromophore
and antiviral activities (Figure 1). These antibiotics have potent DNA biosynthesis from L-tryptophan by unique pathway switching through
binding affinities at a level between nM and mM with different the common intermediate (2S, 3R)-3-hydroxy-L-kynurenine (5).
sequence selectivities. Reflecting these distinct affinities, quinomycin
antibiotics show different inhibitory activities against various RESULTS
enzymes, such as DNA polymerase, RNA polymerase, reverse tran- Bioinformatics analysis of quinomycin family antibiotics
scriptase and topoisomerase.5 Although thiocoraline has promising In the biosynthetic study of echinomycin (1) and triostin, feeding
antitumor activity, most members of this family of compounds are experiments with isotope-labeled amino acids established that the
toxic to mammalian cells.5 To improve the profiles between antitumor QXC moiety was derived from L-tryptophan11 and that the N1 and N4
activity and toxicity, we studied the biosynthetic pathway of 1 and of QXC were derived from the indole and amino groups of
synthesizing its derivatives by genetic engineering. Recently, we have L-tryptophan, respectively.13 Essential genes for echinomycin
achieved de novo production of 1 using E. coli as a heterologous biosynthesis have been identified by de novo production of 1 using
host.6,7 This facilitated synthesis of echinomycin derivatives by che- E. coli as a heterologous host.6 Exclusion of genes for backbone
moenzymatic synthesis with EcmTE8 and by genetic engineering of biosynthesis, modification, regulation and self-resistance leaves six
non-ribosomal peptide synthetase.9,10
genes suggested to be involved in the biosynthesis of QXC. Successful
Quinomycin class peptide antibiotics have characteristic chromo- incorporation of (2S,3S)-[6¢-2H]-3-hydroxy-L-tryptophan (3a)
phores, which are essential components for their biological supported the intermediacy of 3, indicating that b-hydroxylation of
activities by bisintercalation into DNA. Among more than 20 con- tryptophan involves covalent attachment to L-tryptophan to the ACP
geners of this class of antibiotics, only two major chromophores— domain of the non-ribosomal peptide synthetase (Ecm13), hydroxyla-
quinoxaline-2-carboxylic acid (QXC, 6) and 3-hydroxyquinaldic acid tion with cytochrome P-450 monooxygenase (Ecm12), and release of
1Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo, Japan; 2School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan and
3Advanced Science Institute, RIKEN, Wako, Saitama, Japan
Correspondence: Professor H Oikawa, Division of Chemistry, Graduate School of Science, Hokkaido University, Kita-ku Kita 10 Nishi 8, Sapporo, Hokkaido 060-0810, Japan.
E-mail: hoik@sci.hokudai.ac.jp
Dedicated to the late Dr C Richard Hutchinson for his exceptional contributions to natural product biosynthesis, engineering and drug discovery.
Received 2 October 2010; revised 23 October 2010; accepted 25 October 2010; published online 24 November 2010

Biosynthesis of chromophores in quinomycin antibiotics
Y Hirose et al
118
Feeding experiments of (2S,3R)-(6¢-2H)-3-hydroxy-
L-kynurenine (5a)
Me
O
H
N
N
N
O
HN
O
N
H
N
To confirm the intermediacy of 5, feeding experiments with (2S,3R)-
(6¢-2H)-3-hydroxy-L-kynurenine (5a) were used. The synthesis of
5a began with (6¢-2H)-3-hydroxy-L-tryptophan (3a).12 Ozonolysis
of 3a afforded formamide 4a, which was hydrolyzed with trifluoroa-
cetic acid to give 5a in 50% yield (two steps). The resulting labeled
putative precursor 5a was administered to the echinomycin-producing
strain Streptomyces lasaliensis. LC-MS analysis of echinomycin (1) thus
obtained showed significant increases in 1 and 2 Da-shifted peak
intensities at m/z 1102 (d1-MH+) (41%) and 1103 (d2-MH+) (25%)
compared with the natural abundance peak at m/z 1101 (MH+)
(Figure 2a). The data indicated that 5a was incorporated into both
QXC moieties of 1 in a highly efficient manner. Similarly, feeding of 5a
to Streptomyces sp. SNA15896 gave monodeuterated and dideuterated
SW-163D, as evidenced by the observation of the peaks at m/z
1128 (d1-MH+) (22%) and 1129 (d2-MH+) (2%) were increased
(Figure 2b). In this case, only one of the HQA moieties was
incorporated into 2, probably due to significant dilution of the labeled
precursor before the dimerization step of the monomeric tetrapeptide
precursor. These results from feeding experiments supported the
suggestion that 5 is a common intermediate in the biosyntheses of
both QXC and HQA.
O
Me
O
O
O
O
O
S
N
MeS
Me
N
O
O
N
N
N
H
O
NH
O
H
O
Me
echinomycin (1)
Me
O
H
N
N
N
O
HN
O
N
Me
H
N
O
O
O
S
S
O
O
N
N
Me
N
O
N
H
NH
O
N
H
O
Me
triostin A
Me
O
H
OH
N
O
HN
O
N
Me
H
N
O
O
N
O
S
N
MeS
Me
N
Cloning, expression, and purification of Swb1 and Swb2
On the basis of the results of the bioinformatics analyses described
above, we speculated that two gene products, Swb1 (aminotransferase)
and Swb2 (oxidoreductase), were involved in the transformation of
3-hydroxy-L-kynurenine (5) to HQA. Thus, the last two steps are
(1) transamination of 5 and concomitant imine formation giving
3-hydroxykynurenic acid (7); (2) the subsequent reduction and
concomitant dehydration giving HQA (8), as shown in Scheme 1.
To obtain direct support of this proposal, enzymatic conversion of 5–8
was examined.
Both swb1 and swb2 genes were amplified by PCR from genomic
DNA. Amplicons were purified, digested with NdeI/XhoI, and then
inserted into the pET28b vector with incorporation of an N-terminal
His6 tag. The resulting plasmids, pYHSwb1 and pYHSwb2, were used
to transform E. coli BL21 (DE3) and Rosetta strains, respectively. The
recombinant Swb1 and Swb2 were successfully expressed in E. coli
strains at 15 1C as soluble proteins and purified by Ni-chelating
affinity chromatography. SDS-polyacrylamide gel electrophoresis
analysis indicated that the purities of the recombinant proteins are
490 and 470%, respectively (Figure 3).
O
O
N
N
H
O
NH
O
HO
H
O
Me
SW-163D (2)
SMe
S
Me
O
H
OH
H
N
N
HN
S
N
O
Me
O
O
N
S
S
O
O
N
Me
N
O
N
H
NH
O
N
HO
H
O
Me
thiocoraline
Figure 1 Quinomycin family antibiotics.
MeS
the hydroxylated product by the thioesterase (Ecm2),12 as in the case
of tyrosine (novobiocin)16 and histidine (nikkomycin).17 Recently,
four gene clusters of quinomycin-type antibiotics (Figure 1, 1, 2, Enzymatic activity of Swb1 and Swb2, and identification of the
triostin A and thiocoraline) have been identified,6,9,14,15 all of which enzymatic reaction products
share four homologous genes (identity %) (ecm2/trsB/swb14/tioQ Enzyme assay of the putative aminotransferase Swb1 with 5a was
(65–75%), ecm11/trsC/swb10/tioF (30–72%), ecm12/trsB/swb13/tioI conducted in the presence of pyridoxal-5¢-phosphate (pPLP) and
(64–80%), and ecm13/trsR/swb11/tioK (58–76%)), which were pro- a-ketoglutarate. In LC-MS analysis of the reaction products, a novel
posed to be responsible for chromophore biosynthesis. Although the peak was observed on selective ion monitoring at m/z 207. Compar-
clusters of 1 and triostin A consist of homologous genes ecm3/trsP ison of the enzymatic product with a synthetic standard 7 in LC-MS
(80%) and ecm4/trsO (72%) probably responsible for QXC biosynth- analysis, we concluded that the product was identical to 7 (Figure 4a).
esis,6 the clusters of 2 and thiocoraline have alternative unique genes The second enzymatic reaction of the putative oxidoreductase Swb2
swb1/tioG (57%) and swb2/tioH (58%) probably involved in HQA was carried out on 7 in the presence of NADPH. Again in LC-MS
biosynthesis,9 as shown in Scheme 1. Among the four common genes analysis, a new peak appeared at 12.7min on monitoring m/z 190.
for echinomycin chromophore biosynthesis, the putative function This peak was identical to synthetic 8 (Figure 4b). Although Swb1 also
(L-tryptophan 2,3-dioxygenase) of the remaining gene ecm11 sug- converted from kynurenine (9) to kynurenic acid (10) (Figure 4c),
gested that 3-hydroxy-L-kynurenine (5) is a common intermediate in Swb2 did not give any new peaks using 10 as a substrate. These results
the chromophore biosynthesis.
indicated that 5 is an intermediate in the biosynthesis of 8.
The Journal of Antibiotics

Biosynthesis of chromophores in quinomycin antibiotics
Y Hirose et al
119
Ecm13/TrsR/
Swb11/TioK
O
OH
O
Ecm12/TrsB/
Swb13/TioI
R
OH
OH
NH2
NH2
Ecm2/TrsB/
Swb14/TioQ
N
H
N
H
3: R = H
3a: R = D
Ecm11/TrsC/
Swb10/TioF
O
OH
N
N
R
OH
Ecm3/TrsP
Ecm4/TrsO
HO
OH
NH
O
CO2H
O
O
NH2
H2N
6: R = H
6a: R = D
OH
1
OH
R
NH2
OH
O
OH
NHR2
O
NH2
OH
OH
OH
R
OH
R
Swb2/TioH
Swb1/TioG
4: R1 = H, R2 = HCO
4a: R1 = D, R2 = HCO
5: R1, R2 = H
OH
reduction/
dehydration
N
transamination/
cyclization
N
O
O
5a: R1 = D, R2 = H
OH
OH
8: R = H
8a: R = D
7: R = H
7a: R = D
OH
N
H
O
Scheme 1 Proposed biosynthetic pathway of chromophores of echinomycin (1) and SW-163D (2).
Figure 2 ESI-MS spectra of echinomycin (1) and SW-163D (2) from feeding experiments with (2S, 3R)-(6¢-2H)-3-hydroxy-L-kynurenine (5a). (a) (i) non-
labeled 1; (ii) 1 obtained from feeding experiment. (b) (i) non-labeled 2; (ii) 2 obtained from feeding experiment. Assays and HPLC conditions are available
in Experimental section.
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Biosynthesis of chromophores in quinomycin antibiotics
Y Hirose et al
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DISCUSSION
5 was further supported by the results of bioinformatics studies
We performed a feeding experiment with (6¢-2H)-3-hydroxy-L-kynur- indicating that common gene homologs (ecm2/trsB/swb14/tioQ,
enine (5a) to strains producing echinomycin and SW-163D. Efficient ecm11/trsC/swb10/tioF, ecm12/trsB/swb13/tioI and ecm13/trsR/swb11/
incorporation of 5a confirmed its intermediacy in the biosynthesis of tioK) for b-hydroxylation of L-tryptophan and oxidative cleavage of
both chromophores, QXC 6 and HQA 8. Intermediacy of 3 and indole were found in the gene clusters of four quinomycin family
members. In vitro functional analysis of enzymes Swb1 and Swb2
established conversion of 5 to HQA 8.
In a previous study of the biosynthesis of thiocoraline, Salas and
kDa
1
2
kDa
45
1
2
colleagues15 proposed that kynurenine (9) derived from L-tryptophan
with TioF (L-tryptophan-2,3-dioxygenase) and TioL (arylformami-
dase) is an intermediate that is further transformed with TioH
(oxidoreductase) and TioI (cytochrome P-450 monooxygenase)
to HQA 8. Tsodikova and colleagues18 reported functional analysis
of TioF, which converted tryptophan to 9, supporting the interme-
diacy of 9. On the other hand, we unambiguously established enzy-
matic conversion of 5 to HQA 8 with Swb1 (TioG homolog) and
Swb2 (TioH homolog). In addition, our data in the feeding experi-
ment and bioinformatics analysis strongly suggest that cytochrome
P-450 monooxygenase homologs Ecm12/TrsB/Swb13/TioI are involved
in hydroxylation of L-tryptophan, but not that of quinaldic acid.
These data did not fit Salas’ proposed pathway. We speculated that
both 2,3-dioxygenase homologs Swb10 and TioF can catalyze oxida-
tive cleavage of not only tryptophan, but also 3, indicating functional
analysis of TioF is not conclusive evidence on the intermediacy of 9.
Analysis of chromophore biosynthesis can yield insight into one of
the strategies by which microorganisms create novel pathways for
secondary metabolites. Natural peptides often incorporate hydroxy-
lated amino acids.16,17,19,20 In chromophore biosynthesis, the produ-
cing strains utilize similar b-hydroxylation system consisting of
cytochrome P-450, non-ribosomal peptide synthetase, and discrete
thioesterase for conversion of L-tryptophan to 3.16,17 Chromophore
biosynthetic clusters for 1 and 2 consist of genes encoding enzymes for
tryptophan metabolism, such as kynurenic acid21 and nicotinic
45
31
31
21
21
Figure 3 SDS-polyacrylamide gel electrophoresis analysis showing partially
purified Swb1 (a) and Swb2 (b): lane 1, size markers; lane 2, partially
purified Swb1 or Swb2.
5
m/z 226
m/z 206
7
a
b 20
15
10
5
7a
7
m/z 207
m/z 190
8
30
10
20
10
8
m/z 206
m/z 190
60
20
10
5
5
10
15
11
12
13
14
15
(min)
(min)
9
m/z 209
c
10
4
10
m/z 190
20
10
m/z 190
10
10
5
5
10
15
(min)
Figure 4 LC-MS analysis of enzymatic reaction products with Swb1 and Swb2 (ESI-positive mode). (a) (i) the substrate 5; (ii) reaction products with Swb1;
(iii) authentic sample of the product 7. (b) (i) the substrate 7; (ii) reaction products with Swb2; (iii) authentic sample of the product 8. (c) (i) the substrate
9; (ii) reaction products with Swb1; (iii) authentic sample of the product 10. Assays and HPLC conditions are available in Experimental section.
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Biosynthesis of chromophores in quinomycin antibiotics
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Tryptophan metabolism
kynurenine
3-monooxygenase
(Ecm4 homolog)
O
NH2
O
NH2
OH
N
aminotransferase
(Swb1 homolog)
OH
OH
OH
O
O
NH2
NH2
O
OH
L-kynurenine (9)
kynurenic acid (10)
quinolinic acid
nicotinamide
Chromophore biosynthesis
NH2
O
N
OH
OH
1) Ecm4/TrsO
2) Ecm3/TrsP
1) Swb1/TioG
2) Swb2/TioH
OH
OH
N
OH
NH2
O
N
O
O
6
5
8
Scheme 2 Gene homologs found in primary and secondary metabolites.
acid,22,23 which are presumably different from the constitutive (Difco) and 0.4% yeast extract (Difco). All E. coli strains BL21 (DE3)
(Invitrogen, Tokyo, Japan) and Rosetta (Novagen, Tokyo, Japan) in this study
were grown following standard protocols.
enzymes (Scheme 2). After conversion of 3–5 with tryptophan-2,
3-dioxygenase homolog, this common intermediate is transformed
into either 7 with aminotransferase homolog or rearranged product
with monooxygenase homolog (Scheme 1). Final transformations to
chromophores 6 and 8 are catalyzed by specific redox enzymes such as
Ecm3 and Swb2. Therefore, the strategy for creating novel pathways
can be achieved by: (1) incorporation of modification enzymes
transforming a common precursor to an unusual precursor; (2)
duplication of existing genes and mutation to change function and;
(3) recruiting additional enzyme to optimize target structure.
(2S, 3R)-(6¢-2H)-N-formyl-3-hydroxy-L-kynurenine (4a)
A stirred solution of (2S, 3S)-(6¢-2H)-3-hydroxy-L-tryptophan (3a, 100 mg,
0.45mmol) in MeOH (30 ml) was treated with ozone at ꢀ781C for 25min.
Excess dimethylsulfide (1ml, 13.6 mmol) was added, and stirring was con-
tinued to room temperature. The volatile was removed in vacuo and the
resultant residue was purified by HPLC (20% CH3CN/0.1% HCOOH in H2O,
0–10 min, 30%; 10–30min, linear gradient 30–50%; 30–35 min, 50%; UV at
200 nm, 2 mlminꢀ1, 301C) with a column (Inertsil ODS-3, f 3.0250mm,
GL Science) to give 4a (56.4 mg, 53%) along with an identified by-product
(28 mg). 4a: a colorless oil. 1H-NMR (300 MHz, CD3OD)d8.47 (1H, d,
J¼8.3 Hz), 8.38 (1H, s), 7.84 (1H, s), 7.57 (1H, d, J¼8.3 Hz), 5.38 (1H, d,
Although arylformamidase is an essential enzyme for kynurenine
biosynthesis, the corresponding gene is not found in any of the four
quinomycin family gene clusters. Initially, tiol and ecm14 (trsF) were
proposed to catalyze this reaction,6,15 but low-sequence homology and J¼3.4 Hz), 3.99 (1H, d, J¼3.4 Hz); 13C NMR (75 MHz, CD3OD) d 201.6, 170.3,
162.3, 139.0, 134.7, 132.0, 130.2, 125.7, 123.5, 73.2, 58.6; HRMS (ESI) calcd.
for C11H12DN2O5 [M+H]+ 254.0886; found 254.0839; [a]D27 ꢀ14.0 (c 0.5,
MeOH).
absence of the N-formamidase gene in the SW-163 gene cluster
suggested lack of this gene in all quinomycin gene clusters.
In echinomycin biosynthesis, the gene for the aryl-carrier protein
for loading of QXC chromophore is not present on the gene cluster,
but FabC involved in the fatty-acid biosynthesis is acted as an
alternative acyl-carrier protein.24 Therefore, arylformamidase in the
biosynthesis of nicotinic acid pathway may be used as an alternative.
We have already established the de novo synthesis of echinomycin by
introducing three plasmids carrying non-ribosomal peptide synthetase
genes, QXC biosynthetic genes and other auxiliary genes into E. coli.
The findings presented in this paper will lead to switching of the
chromophore from QXC to HQA by exchanging two genes, that is,
ecm3-ecm4 to swb1-swb2. This will add options for diversification of
quinomycin skeleton with engineering of biosynthetic pathways.25,26
(2S 3R)-(6¢-2H)-3-hydroxy-L-kynurenine (5a)
To a stirred solution of amide 4a (56 mg, 0.23mmol) in H2O (10 ml) was
added trifluoroacetic acid (0.5ml, 6.6 mmol) at room temperature. The
mixture was stirred for 1 h and then lyophilized to give a residue, which was
purified by HPLC under the same conditions as 4a to afford 5a (50.5mg, 95%).
5a: an amorphous solid; 1H-NMR (300 MHz, CD3OD) d7.58 (1H, s), 7.25
(1H, d, J¼8.4 Hz), 6.73 (1H, d, J¼8.4 Hz), 5.43 (1H, d, J¼3.1Hz), 4.06 (1H, d,
J¼3.1 Hz); 13C-NMR (75 MHz, CDCl3)d 198.3, 170.6, 135.7, 130.3, 124.4,
122.3, 119.3, 118.3, 70.5, 57.6; HRMS (ESI) calcd. for C10H12DN2O4 [M+H]+
226.0937; found 226.0902; [a]D27 +18.5 (c 0.08, H2O).
Feeding Experiments with (2S, 3R)-(6¢-2H)-3-hydroxy-
L-kynurenine (5a)
EXPERIMENTAL PROCEDURE
Sterile R2YE medium (100 ml) was inoculated with spore suspension (H2O,
5 ml) of Streptomyces sp. SNA15896 from a 7-day R2YE plate. The culture was
incubated at 301C and 130 r.p.m. On the third day after inoculation, the
aqueous solution (5ml) of 20mg of isotopically labeled 5a filtered through
sterilized microfilter (0.22mm) was added into the culture and incubation was
continued for further 4 days. The cells were harvested by centrifugation at
5000 g. The resultant supernatants was extracted with EtOAc. Acetone extracts
of the mycelia were concentrated and extracted with EtOAc. Both extracts from
the supernatants and the mycelia were combined and concentrated in vacuo to
yield the residue, which was dissolved into MeOH (2ml). The extract filtered
with microfilter (0.22 mm) was directly analyzed by LC-ESI-MS (ESI-positive
mode) (ZORBAX XDB-C18, Agilent, f 2.1 50mm); 0.2 mlminꢀ1, 301C,
Chemicals
(2S, 3S)-(6¢-2H)-3-hydroxy-L-tryptophan,12 HQA27 and (2S, 3R)-3-hydroxy-L-
kynurenic acid28 were synthesized as reported previously. All reagents commer-
cially supplied were used as received. Optical rotations were recorded on
JASCO DIP-360 digital polarimeter. NMR spectra were obtained on JEOL ECP-
300 and JEOL a-400 spectrometers for solution of CD3OD. 1H and 13C
chemical shifts were referenced to the solvent signals: 3.30 and 49.0p.p.m.
for CD3OD. Mass spectra were recorded on JEOL JMS-T100CS (ESI).
Flash column chromatography was performed using Kanto Silica Gel 60N.
Bacterial strains, media and culture conditions
Streptomyces lasaliensis ATCC 35851 and Streptomyces sp. SNA15896 were solvent system, CH3CN/0.1% TFA in H2O, 0–5 min, 20%; 5–25 min, linear
grown at 30 1C on R2YE medium containing 0.4% maltose, 1.0% malt extract gradient 20–100%; 25–30min, 100%.
The Journal of Antibiotics

Biosynthesis of chromophores in quinomycin antibiotics
Y Hirose et al
122
was supported by JSPS Grants-in-Aid for Scientific Research (B) 20310126 and
by MEXT research grant on innovative area 22108002 to H Oikawa.
Similar feeding experiment was used to S. lasaliensis in the essentially same
manner. Labeled precursor was fed on the second day after inoculation and
incubation was continued for further 4 days.
Cloning, expression and purification of Swb1
The swb1 gene was obtained by PCR amplification with SP-taq polymerase
(LaboPass) using a genomic DNA from Streptomyces sp. SNA15896. Primers for
swb1 (swb1-Fw: 5¢-AAAGAGAACCATATGGCAGCATCGGCGCCAG-3¢ and
swb1-Rv: 5¢-TTTGAGCTCGATGGCGACGCGCCGCTT-3¢) contained NdeI
and XhoI sites (underlined, restriction endonuclease site) and the amplified
gene was cloned into the corresponding sites of pET28b vector (Novagen) to
yield pYHSwb1 for the production of Swb1. E. coli. BL21 (DE3) transformed
with pYHSwb1 was incubated at 37 1C overnight in 10 ml LB medium
supplemented with kanamycin (100mg mlꢀ1). Each liter of fresh LB medium
with the same antibiotics at the same concentration as described above was
inoculated with 5 ml of the overnight culture and incubated at 371C until the
optical density at 600 nm reached 0.6. Then, expression of swb1 gene was
induced with 100 mM isopropyl-b-D-thiogalactopyranoside (IPTG) at 151C.
Incubation was continued for another 24h, after which cells were harvested by
centrifugation at 2500 g. All subsequent procedures were performed at 4 1C or
on ice. Harvested cells were resuspended in disruption buffer (0.1M Tris-HCl
(pH 7.2) and 20% (v/v) glycerol). After addition of PLP (a final concentration
10 mM), cells were disrupted by a French Press cell, and the lysate was clarified by
centrifugation at 18600g. The soluble fraction containing protein was applied to
a Ni-NTA affinity column (GE Healthcare, Tokyo, Japan) previously equilibrated
with binding buffer (0.1 M Tris-HCl (pH 7.2), 10 mM PLP and 20% (v/v) glycerol)
supplemented with 10 mM imidazole, at a flow rate of 1 ml minꢀ1. The column
was washed with the binding buffer with 10 mM imidazole, then proteins were
eluted with a gradient of 10–500 mM imidazole over 100 ml of the binding buffer.
Fractions containing protein with target molecular weight were pooled. Buffer
exchange into the reaction buffer (100 mM Tris-HCl, 20% (v/v) glycerol, pH 7.2)
was carried out using a PD-10 column (GE Healthcare). Protein concentration
was estimated using the Bio-Rad protein assay kit (BioRad, Tokyo, Japan) with
bovine immunoglobulin G as a standard. Purified protein samples were analyzed
by SDS-polyacrylamide gel electrophoresis.
1
2
Dell, A. W. et al. Structure revision of the antibiotic echinomycin. J. Am. Chem. Soc.
97, 2497 (1975).
Kurosawa, K., Takahashi, K. & Tsuda, E. SW-163C and E, novel antitumor depsipep-
tides produced by Streptomyces sp. I. Taxonomy, fermentation, isolation and biological
activities. J. Antibiot. 54, 615–621 (2001).
Takahashi, K., Koshino, H., Esumi, Y., Tsuda, E. & Kurosawa, K. SW-163C and E, novel
antitumor depsipeptides produced by Streptomyces sp. II. Structure elucidation.
J. Antibiot. 54, 622–627 (2001).
Nakaya, M. et al. Relative and absolute configuration of antitumor agent SW-163D.
Biosci. Biotechnol. Biochem. 71, 2969–2976 (2007).
Dawson, S., Malkinson, J. P., Paumier, D. & Searcey, M. Bisintercalator natural
products with potential therapeutic applications: isolation, structure determination,
synthetic and biological studies. Nat. Prod. Rep. 24, 109–126 (2007).
Watanabe, K. et al. Total biosynthesis of antitumor nonribosomal peptides in Escher-
ichia coli. Nat. Chem. Biol. 2, 423–428 (2006).
Watanabe, K. & Oikawa, H. Robust platform for de novo production of heterologous
polyketides and nonribosomal peptides in Escherichia coli. Org. Biomol. Chem. 5,
593–602 (2007).
Koketsu, K., Oguri, H., Watanabe, K. & Oikawa, H. Enzymatic macrolactonization in the
presence of DNA leading to triostin A analogs. Chem. Biol. 15, 818–828 (2008).
Watanabe, K. et al. Rationally engineered total biosynthesis of a synthetic analogue
of a natural quinomycin depsipeptide in Escherichia coli. Chem. Bio. Chem. 10,
1965–1968 (2009).
3
4
5
6
7
8
9
10 Watanabe, K. et al. Escherichia coli allows efficient modular incorporation of newly
isolated quinomycin biosynthetic enzyme into echinomycin biosynthetic pathway for
rational design and synthesis of potent antibiotic unnatural natural product. J. Am.
Chem. Soc. 131, 9347–9353 (2009).
11 Yoshida, T., Katagiri, K. & Yokozawa, S. Studies on quinoxaline antibiotics. II. Isolation
and properties of quinomycins A, B, C. J. Antibiot. Ser. A 14, 330–334 (1961).
12 Koketsu, K., Oguri, H., Watanabe, K. & Oikawa, H. Identification and stereochemical
assignment of the b-hydroxytryptophan intermediate in the echinomycin biosynthetic
pathway 8, 4719–4722 (2006).
13 Reid, D. G., Doddrell, D. M., Williams, D. H. & Fox, K. R. A 15N nuclear magnetic
resonance study of the biosynthesis of quinoxaline antibiotics. Biochim. Biophys.
Acta 798, 111–114 (1984).
14 Praseuth, A. P. et al. Complete sequence of biosynthetic gene cluster responsible for
producing triostin A and evaluation of quinomycin-type antibiotics from Streptomyces
triostinicus. Biotechnol. Prog. 24, 1226–1231 (2008).
15 Lombo, F. et al. Deciphering the biosynthesis pathway of the antitumor thiocoraline
Cloning, expression and purification of Swb2
from
a marine actinomycete and its expression in two streptomyces species.
The swb2 gene was obtained by PCR amplification with primers (swb2-Fw:
5¢–AAAAGTGCGCATATGAGTGTGTCCGGCAAG-3¢ and swb2-Rv: 5¢-TTT
GAGCTCGCAGCAAGACGGCGG-3¢) contained NdeI and XhoI sites (under-
lined, restriction endonuclease site) and the amplified gene was cloned into the
corresponding sites of pET28b vector (Novagen) to yield pYHSwb2 for the
production of Swb2. The procedure from transformation to purification of
Swb2 was same as that of Swb1 except use of E. coli (DE3) Rosetta cell in
transformation and absence of PLP in the buffers during purification.
ChemBioChem 7, 366–376 (2006).
16 Chen, H. & Walsh, C. T. Coumarin formation in novobiocin biosynthesis: beta-hydro-
xylation of the aminoacyl enzyme tyrosyl-S-NovH by a cytochrome P450 NovI. Chem.
Biol. 8, 301–312 (2001).
17 Chen, H., Hubbard, B. K., O’Connor, S. E. & Walsh, C. T. Formation of beta-hydroxy
histidine in the biosynthesis of nikkomycin antibiotics. Chem. Biol. 9, 103–112 (2002).
18 Sheoran, A., King, A., Velasco, A., Pero, J. M. & Garneau-Tsodikova, S. Characterization
of TioF, a tryptophan 2,3-dioxygenase involved in 3-hydroxyquinaldic acid formation
during thiocoraline biosynthesis. Mol. Biosyst. 4, 622–628 (2008).
19 Yin, X. H. & Zabriskie, T. M. VioC is a non-heme iron, alpha-ketoglutarate-dependent
oxygenase that catalyzes the formation of 3S-hydroxy-L-arginine during viomycin
biosynthesis. Chem. Bio. Chem. 5, 1274–1277 (2004).
Enzymatic reactions with Swb1 and Swb2
20 Strieker, M., Kopp, F., Mahlert, C., Essen, L. O. & Marahiel, M. A. Mechanistic and
structural basis of stereospecific C beta-hydroxylation in calcium-dependent antibiotic,
a daptomycin-type lipopeptide. ACS Chem. Biol. 2, 187–196 (2007).
21 Han, Q., Fang, J. M. & Li, J. Y. Kynurenine aminotransferase and glutamine transami-
nase K of Escherichia coli: identity with aspartate aminotransferase. Biochem. J. 360,
617–623 (2001).
22 Kurnasov, O. et al. NAD biosynthesis: identification of the tryptophan to quinolinate
pathway in bacteria. Chem. Biol. 10, 1195–1204 (2003).
23 Lima, W. C., Varani, A. M. & Menck, C. F. M. NAD biosynthesis evolution in bacteria:
lateral gene transfer of kynurenine pathway in Xanthomonadales and Flavobacteriales.
Mol. Biol. Evol. 26, 399–406 (2008).
24 Schmoock, G. et al. Functional cross-talk between fatty acid synthesis and
nonribosomal peptide synthesis in quinoxaline antibiotic-producing streptomycetes.
J. Biol. Chem. 280, 4339–4349 (2005).
25 Watanabe, K., Oguri, H. & Oikawa, H. Enzymatic synthesis of molecular skeletons of
complex antitumor antibiotics with non-ribosomal peptide synthetases. J. Synth. Org.
Chem., Jpn. 67, 1152–1160 (2009).
26 Watanabe, K., Oguri, H. & Oikawa, H. Diversification of echinomycin molecular structure
by way of chemoenzymatic synthesis and heterologous expression of the engineered
echinomycin biosynthetic pathway. Curr. Opin. Chem. Biol. 13, 189–196 (2009).
27 Riego, E., Bayo, N., Cuevas, C., Albericio, F. & Alvarez, M. A new approach to
3-hydroxyquinoline-2-carboxylic acid. Tetrahedron 61, 1407–1411 (2005).
28 Behrman, E. J., Kiser, R. L., Garas, W. F., Behrman, E. C. & Pitt, B. M. Conversion of
4-quinolones into 3-hydroxy-4-quinolones via the corresponding sulfates. J. Chem.
Res. -S 164–165 (1995).
Swb1 assay was performed in a total volume of 100ml in Tris-HCl buffer
(100mM, pH 7.2) with 14mM Swb1, 10mM PLP and 100 mM a-ketoglutarate at
30 1C for 12 h. The substrate concentration was 100 mM for 3-hydroxy-L-
kynurenine (5) and L-kynurenine. Reactions were terminated by addition of
MeOH (100ml), and the resultant mixture was vortexed and centrifugated at
13 000 g for 2 min. After concentration of the supernatant using centrifugal
evaporator, the residue was dissolved with water (100 ml). The reaction
products were analyzed by LC-ESI-MS (ESI-positive mode) (Wakosil II
5C18, Wako, Tokyo, Japan) f 2.1 50mm); 0.2 mlminꢀ1, 301C, solvent system,
CH3CN/0.1% TFA in H2O, 0–5 min, 0%; 5–15min, linear gradient 0–100%;
15–20 min, 100%. Swb2 assays were performed in a total volume of 100 ml in
Tris-HCl buffer (100mM, pH 7.2) with 7 mM Swb2 and 100 mM NADPH at 30 1C
for 12 h. The substrate concentration was 100mM for 3-hydroxykynurenic acid
(7) and kynurenic acid. Work-up and analytical conditions were performed in
the same manner as in the experiment with Swb1.
ACKNOWLEDGEMENTS
We are indebted to Dr Tetsuo Tokiwano for preparation of 3-hydroxyquinaldic
acid and to Dr N Washida, Daiichi-Sankyou Pharmaceutical Co. Ltd. for
providing the SW-163 producing strain, Streptomyces sp. SNA15896. This work
The Journal of Antibiotics