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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, César Sánchez, Ben Shen
DOI: http://dx.doi.org/10.1111/j.1574-6968.2000.tb09225.x 171-175 First published online: 1 August 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.

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 modifications [1]. A typical NRPS module consists of an adenylation (A) domain for amino acid recognition and activation, a peptidyl carrier protein (PCP) for thioesterification 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 modified, and these modifications are catalyzed by additional domains, such as epimerization (E) [1], cyclization (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 five-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 defined (Fig. 1B).

Figure 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.

We have previously proposed that the NRPS-2, NRPS-1 (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 identification of an oxidation (Ox) domain within blmIII, overexpressions of the blmIII-Ox domain and blmIII gene in Escherichia coli, and the purification and characterization of the BlmIII-Ox and BlmIII proteins. Our results established that the BlmIII-Ox 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 amplified from the blm gene cluster [7,8] (GenBank accession number AF210249) by PCR using a forward primer of 5′-ATATGGATCCACGAGCGCCCGGCCCACGCCGACA-3′ (the BamHI site is underlined) and a reverse primer of 5′-ATTGTCGACTCACGTGCCGGTTCACGGGGCCTC-3′ (the SalI site is underlined). The PCR-amplified 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 amplified by PCR using a forward primer of 5′-GTCAAGCTTGTGATGGTCGGCCGCCACCTC-3′ (the BamHI site is underlined) and the same reverse primer as for blmIII. The PCR-amplified 1014-bp BamHI/SalI fragment was cloned into pET28a to yield pBS16. The fidelity of the PCR products was confirmed by sequencing. Expression of blmIII in E. coli BL21(DE-3)(pBS15) or blmIII-Ox in E. coli BL21(DE-3)(pBS16) and purification of BlmIII or BlmIII-Ox by affinity chromatography on Ni-NTA resin were carried out under the conditions recommended by the manufacturer (Novagen). The purified 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 −80°C 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].

Figure 2

(A) Sequence comparison between the BlmIII-Ox domain and a group of oxidoreductases. Only the regions that share significant 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 defined in the text.

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 exemplified 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 or serine side chain onto the preceding carbonyl group of the premicrocin B17 substrate to afford 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 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 first 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 thiazole-containing 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 confirm 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, defining 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 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 [16], Ox is the first 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 purified BlmIII-Ox to homogeneity. The purified 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 flavin (Fig. 4A). This flavin 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].

Figure 3

Overexpression in E. coli and purification of BlmIII-Ox (A) and BlmIII (B). Lane 1, molecular mass markers; lane 2, total soluble proteins; lane 3, purified BlmIII-Ox or BlmIII.

Figure 4

Prosthetic group identification 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 flavin prosthetic group dissociated from BlmOx, and (D) a mixture of samples (B) and (C).

Since the growing peptide intermediates are covalently 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)-4-thiazolecarboxylic 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 purified 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 purified 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 confirmed 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 identified 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 thiazole-forming 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.

Acknowledgments

We thank Dr. Christopher T. Walsh, Harvard Medical School, Boston, for samples of HPT-COOH and HPT-Cys. 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.

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