FEMS Microbiology Letters 189 (2000) 171^175
www.fems-microbiology.org
An oxidation domain in the BlmIII non-ribosomal peptide
synthetase probably catalyzing thiazole formation in the biosynthesis
of the anti-tumor drug bleomycin in Streptomyces verticillus
ATCC15003
Liangcheng Du, Mei Chen, Cësar Sänchez, Ben Shen *
Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA
Received 17 March 2000 ; received in revised form 5 June 2000; accepted 12 June 2000
Abstract
We have previously proposed that the BlmIV and BlmIII non-ribosomal peptide synthetases are involved in the formation of the
bithiazole moiety of the anti-tumor drug bleomycin in Streptomyces verticillus ATCC15003. We report here the identification and
characterization of an oxidation domain in BlmIII. The oxidation domain shows local homology to a family of oxidoreductases and is
present in all thiazole-forming non-ribosomal peptide synthetase modules known to date. Both the blmIII-Ox domain and blmIII gene were
expressed in Escherichia coli, and the resulting BlmIII-Ox and BlmIII proteins were purified to homogeneity. The oxidation domain
contains one molar equivalent of non-covalently bound FMN as a prosthetic group. These results provide experimental evidence for an
oxidation domain within non-ribosomal peptide synthetases, suggesting that BlmIII-Ox probably catalyzes the thiazoline to thiazole
oxidation in bleomycin biosynthesis. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All
rights reserved.
Keywords : Bleomycin; Biosynthesis ; Non-ribosomal peptide synthetase; Oxidation domain ; Thiazole; Streptomyces verticillus
1. Introduction
Many non-ribosomal peptides are clinically important
drugs, such as cyclosporins, bleomycin (BLM), vancomycins and penicillins. They are synthesized from amino
acids by the non-ribosomal peptide synthetases (NRPSs).
NRPSs have a modular structure, and each module consists of distinctive domains catalyzing all the steps essential
for one cycle of peptide elongation and associated modi¢cations [1]. A typical NRPS module consists of an adenylation (A) domain for amino acid recognition and activation, a peptidyl carrier protein (PCP) for
thioesteri¢cation of the activated amino acid, and a condensation (C) domain for transpeptidation between the
adjacent peptidyl and amino acyl thioesters [1,2]. The nascent amino acyl or peptidyl intermediates can be further
modi¢ed, and these modi¢cations are catalyzed by additional domains, such as epimerization (E) [1], cyclization
* Corresponding author. Tel. : +1 (530) 754-9382;
Fax: +1 (530) 752-8995; E-mail: shen@chem.ucdavis.edu
(Cy) [3,4], N-methylation (MT) [1], monooxygenation
(MonoOx) [5], reduction (R) [6] or thioesterase (TE) domains [1]. Novel domains are continuously emerging as
new gene clusters for peptide biosynthesis are being characterized.
The ¢ve-member heterocycles, such as thiazolidine, thiazoline and thiazole, are common structural features of
many sulfur-containing peptides (Fig. 1A). These heterocycles result from heterocyclization of the cysteine side
chain onto the preceding carbonyl group of the peptide
substrate to yield a thiazoline, catalyzed by the Cy domain
of an NRPS [3,4]. However, the subsequent thiazoline to
thiazolidine or thizoline to thiazole conversion requires an
additional reduction or oxidation step, respectively, domains catalyzing either of which are yet to be de¢ned
(Fig. 1B).
We have previously proposed that the NRPS-2, NRPS1 (encoded by blmIV) and NRPS-0 modules (encoded by
blmIII) are involved in the biosynthesis of the bithiazole
moiety of BLM in Streptomyces verticillus ATCC15003
[7,8]. We report here the identi¢cation of an oxidation
(Ox) domain within blmIII, overexpressions of the
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 2 7 4 - 3
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L. Du et al. / FEMS Microbiology Letters 189 (2000) 171^175
blmIII-Ox domain and blmIII gene in Escherichia coli, and
the puri¢cation and characterization of the BlmIII-Ox and
BlmIII proteins. Our results established that the BlmIIIOx domain contains one molar equivalent of non-covalently bound FMN, supporting its role in catalyzing thiazole biosynthesis.
2. Materials and methods
The blmIII gene, encoding an inactive A, a PCP and an
Ox domain (Fig. 2C), was ampli¢ed from the blm gene
cluster [7,8] (GenBank accession number AF210249) by
PCR using a forward primer of 5P-ATATGGATCCACGAGCGCCCGGCCCACGCCGACA-3P (the BamHI site
is underlined) and a reverse primer of 5P-ATTGTCGACTCACGTGCCGGTTCACGGGGCCTC-3P (the SalI site is
underlined). The PCR-ampli¢ed 2814-bp BamHI/SalI
fragment was cloned into the same sites of pET28a (Novagen, Madison, WI, USA) to yield pBS15. The blmIII-Ox
alone was similarly ampli¢ed by PCR using a forward
primer of 5P-GTCAAGCTTGTGATGGTCGGCCGCCACCTC-3P (the BamHI site is underlined) and the
same reverse primer as for blmIII. The PCR-ampli¢ed
1014-bp BamHI/SalI fragment was cloned into pET28a
to yield pBS16. The ¢delity of the PCR products was
con¢rmed by sequencing. Expression of blmIII in E. coli
BL21(DE-3)(pBS15) or blmIII-Ox in E. coli BL21(DE3)(pBS16) and puri¢cation of BlmIII or BlmIII-Ox by
a¤nity chromatography on Ni-NTA resin were carried
out under the conditions recommended by the manufacturer (Novagen). The puri¢ed proteins were desalted and
stored in 50 mM Tris^HCl, pH 7.2, 10 mM MgCl2 , 1 mM
EDTA, 2 mM dithiothreitol, 10% glycerol at 380³C for in
vitro assay. The prosthetic group of BlmIII-Ox was determined by spectroscopic and high performance liquid chromatography (HPLC) analyses as described previously [9].
3. Results and discussion
Five-member heterocycles are common structures for
many natural products, such as thiazolidine in yersiniabactin, thiazoline in anguibactin, bacitracin and yersiniabactin, and thiazole in BLM, epothilone, leinamycin and myxothiazol (Fig. 1A). Two mechanisms are known for the
biosynthesis of these heterocycles from a peptide precursor. One is exempli¢ed by microcin B17 biosynthesis,
where the heterocycle-forming steps occur post-translationally [10], and the other emerges from non-ribosomal
peptide biosynthesis, where the peptide elongation and
heterocycle-forming steps proceed processively [3,4]. The
formation of the oxazoles and thiazoles in microcin B17 is
catalyzed by the microcin B17 synthase complex that consists of three discrete proteins, McbBCD [10]. McbB is a
cyclase catalyzing heterocyclization of either the cysteine
Fig. 1. (A) Examples of natural products containing thiazolidine, thiazoline or thiazole rings (shaded) and (B) a proposed general mechanism
for thiazoline, thiazolidine and thiazole biosynthesis.
or serine side chain onto the preceding carbonyl group of
the premicrocin B17 substrate to a¡ord the oxazoline or
thiazoline intermediates. McbC is an FMN-containing
oxidoreductase catalyzing subsequent oxazoline/thiazoline
to oxazole/thiazole oxidations. In functional analogy to
the McbB cyclase, the Cy domain of an NRPS catalyzes
the similar cyclization step, yielding the thiazoline moiety
found in non-ribosomal peptides (Fig. 1B) [4]. However, it
is not known how an NRPS oxidizes a thiazoline into a
thiazole.
The anti-tumor antibiotic BLM is characterized by a
bithiazole structure (Fig. 1A). We have cloned the blm
gene cluster from S. verticillus and proposed that BlmIV
and BlmIII are involved in the formation of the bithiazole
moiety on the basis that both NRPS-1 and NRPS-0 contain a Cy domain (Fig. 2C) [7,8]. However, to convert a
thiazoline into a thiazole apparently requires an additional
oxidation domain ^ an McbC homolog, in functional
analogy to the microcin B17 synthase. Impelled by the
latter hypothesis, we found a previously unknown region
at the C-terminus of BlmIII that shows local similarities,
designated Ox-1 and Ox-2, to a group of putative or
known oxidoreductases, including McbC (Fig. 2A). On
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173
Fig. 2. (A) Sequence comparison between the BlmIII-Ox domain and a group of oxidoreductases. Only the regions that share signi¢cant homology are
shown. The conserved Ox-1 and Ox-2 motifs are underlined. The numbers for each sequence indicate the positions of amino acid residues in respective
proteins. (B) Sequence comparison among Ox domains found in NRPSs. The Ox-1 and Ox-2 motifs are underlined to indicate their locations in reference to the oxidoreductases. The numbers for each Ox domain refer to the positions of amino acid residues in respective NRPS proteins. (C) Domain
organization of thiazole-forming NRPS modules of BlmIV and BlmIII from the BLM cluster [8], of EposP from the epothilone cluster [11,12], and of
MtaC and MtaD from the myxothiazol cluster [5]. The Ox domains are shaded to emphasize their unique location and other abbreviations are de¢ned
in the text.
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L. Du et al. / FEMS Microbiology Letters 189 (2000) 171^175
the basis of sequence homology to these oxidoreductases,
especially to McbC, we named this region as an Ox domain and propose that the BlmIII-Ox domain is responsible for the formation of the second thiazole ring in BLM
biosynthesis by catalyzing a thiazoline to thiazole oxidation. (The formation of the ¢rst thiazole ring in BLM
biosynthesis has previously been proposed to be catalyzed
by a discrete oxidase, ORF8, consistent with the lack of an
Ox domain in BlmIV [7,8]).
Two additional biosynthesis gene clusters for thiazolecontaining peptides, epothilone [11,12] and myxothiazol
[5], have been characterized recently. While a similar Ox
domain was suggested by Molnar et al. from the epothilone gene cluster [11], Ox domain was not reported by
Silakowski et al. from the myxothiazol gene cluster [5]
(it was suggested under GenBank accession number
AF188287). Recognizing the common structural feature
of a thiazole or a bithiazole shared by BLM, epothilone
and myxothiazol, we indeed found two Ox domains in the
unknown regions of mtaC and mtaD within the myxothiazol gene cluster [5], in addition to con¢rm the previously
proposed Ox domain within the epothilone gene cluster
[11,12]. (While this manuscript is in revision, Julien et al.
reported the sequence analysis of the epothilone gene cluster, de¢ning a similar Ox domain along with the two Ox
domains from mtaCD [13]). These Ox domains are highly
homologous, exhibiting around 40% identity and 50% similarity among each other, and the afore-mentioned Ox-1
and Ox-2 motifs are among the most conserved regions in
these domains (Fig. 2B). TfxB is an McbC homolog involved in the biosynthesis of antibiotic trifolitoxin [14],
and MJ1384 (accession number AAB99394) and MJ1384
(accession number AAB84622) are hypothetic proteins
proposed to be NADH oxidases. The Ox domain is
present only in the thiazole-forming NRPS modules, but
not in the thiazolidine- or thiazoline-forming NRPS modules, such as those for bacitracin [3], yersiniabactin [4] and
anguibactin [15], consistent with the fact that thiazolidine
or thiazoline biosynthesis does not require an Ox domain.
Noteworthy is the localization of the Ox domain within
an NRPS module (Fig. 2C). In BlmIII and MtaC, the Ox
Fig. 3. Overexpression in E. coli and puri¢cation of BlmIII-Ox (A) and
BlmIII (B). Lane 1, molecular mass markers ; lane 2, total soluble proteins ; lane 3, puri¢ed BlmIII-Ox or BlmIII.
Fig. 4. Prosthetic group identi¢cation for the BlmIII-Ox domain. (A)
UV-Vis spectrum of BlmIII-Ox. HPLC analysis of (B) a mixture of
pure FAD and FMN, (C) the £avin prosthetic group dissociated from
BlmOx, and (D) a mixture of samples (B) and (C).
domain is located downstream of the PCP domain, whereas in EposP and MtaD, the Ox domain is located between
the A8 and A9 motifs of an A domain. In contrast to all
other NRPS domains known to date whose relative position in a given NRPS module is highly conserved [1^6], Ox
is the ¢rst domain with two possible locations within an
NRPS module.
To provide experimental evidence for its catalytic role in
thiazole biosynthesis, we overexpressed the blmIII-Ox domain in E. coli and puri¢ed BlmIII-Ox to homogeneity.
The puri¢ed protein migrated with a Mr of 55 000 (Fig.
3A), which is larger than that of 38 074, calculated according to the blmIII-Ox domain plus the N-terminal His6 -tag.
(It is not known why BlmIII-Ox migrated abnormally
under SDS^polyacrylamide gel electrophoresis. The same
abnormal behavior was also observed for BlmIII. While
the calculated Mr for BlmIII, according to blmIII plus the
N-terminal His6 -tag, is 101 020, BlmIII migrated with a
Mr of 120 000 (Fig. 3B)). The UV-Vis spectrum of
BlmIII-Ox exhibits the characteristic absorption maxima
at 375 and 450 nm for the presence of a £avin (Fig. 4A).
This £avin prosthetic group was subsequently determined
by HPLC to be FMN (Fig. 4B^D), and the molar ratio of
BlmIII-Ox/FMN is 1:1. These results are consistent with
the sequence-based prediction for BlmIII-Ox^McbC has
been shown to contain a non-covalently bound FMN
prosthetic group [10].
Since the growing peptide intermediates are covalently
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linked to the NRPS complex via the PCP domains during
the elongation process [1], the likely substrate for the
BlmIII-Ox domain would be the enzyme-bound growing
peptide-polyketide intermediate, which is not readily available. In the absence of the native substrate, we attempted
to demonstrate the enzyme activity of the BlmIII-Ox domain in vitro using 4,5-dihydro-2-(2-hydroxyphenyl)-4thiazolecarboxylic acid (HPT-COOH) and N-{[2-(2-hydroxyphenyl)]-4,5-dihydrothiazole-4-carbonyl}-cysteine
(HPT-Cys) as substrate mimics [4]. Both HPT-COOH and
HPT-Cys were incubated in the presence of the puri¢ed
BlmIII-Ox, with or without the addition of NAD‡ ,
NADP‡ , ATP, Zn2‡ or Ca2‡ , and the reaction products
were subjected to HPLC and mass spectral analyses. However, no oxidation was observed under any of the conditions examined, despite the fact that both HPT-COOH
and HPT-Cys contain a thiazoline ring. To exclude the
possibility that the BlmIII-Ox domain may be inactive
when produced alone, we expressed blmIII in E. coli and
puri¢ed the intact BlmIII protein (Fig. 3B). BlmIII was
also inactive in catalyzing HPT-COOH or HPT-Cys oxidation under any of the conditions tested, although the
integrity of BlmIII was con¢rmed by assaying its abilities
to be phosphopantatheinylated at the PCP domain, followed by acylation of the resultant holo-PCP with the
NRPS-1 activated cysteine in vitro (data not shown) [1].
At this stage, we have to conclude that simple thiazolines,
such as HPT-COOH and HPT-Cys, cannot act as substrate mimics for the BlmIII-Ox domain.
In summary, we have identi¢ed an Ox domain in the
BlmIII NRPS. The BlmIII-Ox domain shows local homology to a family of oxidoreductases and contains one molar
equivalent of non-covalently bound FMN as a prosthetic
group. Similar Ox domain is present only in the thiazoleforming NRPS modules. These results provide experimental evidence for the Ox domain within NRPSs, suggesting
that BlmIII-Ox probably catalyzes the thiazoline to thiazole oxidation in BLM biosynthesis.
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
Acknowledgements
[13]
We thank Dr. Christopher T. Walsh, Harvard Medical
School, Boston, for samples of HPT-COOH and HPTCys. This work was supported in part by an Institutional
Research Grant from the American Cancer Society and
the School of Medicine, University of California, Davis,
the NIH Grant AI40475, and the Searle Scholars Program/The Chicago Community Trust.
[14]
[15]
175
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