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Antimicrobial Agents and Chemotherapy, April 2006, p. 1136-1142, Vol. 50, No. 4
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.4.1136-1142.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Structure-Activity Relationships of Aminocoumarin-Type Gyrase and Topoisomerase IV Inhibitors Obtained by Combinatorial Biosynthesis

Ruth H. Flatman,1 Alessandra Eustaquio,2 Shu-Ming Li,2 Lutz Heide,2 and Anthony Maxwell1*

Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, United Kingdom,1 Pharmazeutische Biologie, Pharmazeutisches Institut, Auf der Morgenstelle 8, D-72076 Tübingen, Germany2

Received 26 October 2005/ Returned for modification 26 November 2005/ Accepted 4 January 2006


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ABSTRACT
 
Novobiocin and clorobiocin are gyrase inhibitors produced by Streptomyces strains. Structurally, the two compounds differ only by substitution at two positions: CH3 versus Cl at position 8' of the aminocoumarin ring and carbamoyl versus 5-methyl-pyrrol-2-carbonyl (MePC) at the 3"-OH of noviose. Using genetic engineering, we generated a series of analogs carrying H, CH3, or Cl at 8' and H, carbamoyl, or MePC at 3"-OH. Comparison of the gyrase inhibitory activities of all nine structural permutations confirmed that acylation of 3"-OH is essential for activity, with MePC being more effective than carbamoyl. Substitution at 8' further enhanced activity, but the effect of CH3 or Cl depended on the nature of the acyl group at 3": in the presence of carbamoyl at 3", CH3 resulted in higher activity; in the presence of MePC at 3", Cl resulted in higher activity. This suggests that the structures of both natural compounds are highly evolved for optimal interaction with gyrase. In a second series of experiments, clorobiocin derivatives with and without the methyl group at 4"-OH of noviose, and with different positions of the MePC group of noviose, were tested. Again clorobiocin was superior to all of its analogs. The activities of all compounds were also tested against topoisomerase IV (topo IV). Clorobiocin stood out as a remarkably effective topo IV inhibitor. The relative activities of the different compounds toward topo IV showed a pattern similar to that of the relative gyrase-inhibitory activities. This is the first report of a systematic evaluation of a series of aminocoumarins against both gyrase and topo IV. The results give further insight into the structure-activity relationships of aminocoumarin antibiotics.


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INTRODUCTION
 
Antibiotic resistance is a major problem worldwide, and it is therefore essential that new antibacterial drugs be developed. DNA gyrase and DNA topoisomerase IV (topo IV) are important targets for antibacterial drugs (1, 14, 16). Topo IV is important for unlinking daughter chromosomes during DNA replication, and gyrase is important in relaxing the supercoils that accumulate ahead of replication forks and transcription complexes (10). Both enzymes are capable of relaxing DNA, but gyrase is unique in being able to introduce negative supercoils; topo IV is a very efficient DNA decatenase. Both enzymes are ATP dependent.

Gyrase and topo IV are type II topoisomerases, which means that they catalyze the cleavage of both strands of the DNA duplex. They comprise A2B2 heterotetramers and share significant sequence similarity. Their general mechanism involves cleavage of both DNA strands and the passage of a segment of DNA through the break before resealing of the DNA. The involvement of transient DNA cleavage during their mechanism of action reveals a vulnerability that can be exploited by antibacterial agents that stabilize the cleavage complex, such as the fluoroquinolones (1). However, there are other compounds that inhibit these enzymes via binding to their ATP-binding sites (16).

One of the main classes of antibiotics that target gyrase and topo IV is the aminocoumarins. Clorobiocin and novobiocin belong to this group, and both contain the characteristic 3-amino-4,7-dihydroxycoumarin core linked at the 7-OH group to a noviose sugar moiety and at the 3-NH2 group to a prenylated 4-hydroxybenzoyl moiety. Clorobiocin and novobiocin are very potent inhibitors of gyrase that act by inhibiting ATP hydrolysis (16); they also inhibit the ATPase reaction of topo IV. However, their clinical use is limited due to their poor solubility and toxic side effects. The interaction mechanism is well characterized for both compounds and includes structural information from crystal complexes of both aminocoumarins with the N-terminal subdomain of GyrB (8, 11, 16, 23). These structures showed that there is overlap between the binding sites of the coumarins and ATP; the adenine ring of ATP binds at the same place as the noviose sugar. This explains the competitive nature of aminocoumarin inhibition. The most frequently occurring single point mutations in Escherichia coli that confer resistance to aminocoumarins are at Arg136, and this amino acid residue has been found to form a key hydrogen bond with the coumarin ring.

Structurally, novobiocin and clorobiocin differ only by the substituents in two positions. Novobiocin carries a methyl group at position 8' of the aminocoumarin ring and a carbamoyl group at 3"-OH of noviose. In contrast, clorobiocin contains a chlorine atom at 8' and a 5-methyl-pyrrol-2-carbonyl (MePC) moiety at 3" (Fig. 1). When novobiocin binds to gyrase, the carbamoyl group forms a hydrogen bond with an ordered water molecule in the ATP-binding site. In contrast, the larger MePC group of clorobiocin displaces two ordered water molecules from the ATP-binding site (9, 17, 23).


Figure 1
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  		FIG. 1. Series 1 of novobiocin and clorobiocin analogs and their inhibitory effects on DNA gyrase and topo IV. The concentrations of novclobiocins that cause 50% inhibition of gyrase supercoiling and topo IV decatenation are given. IC50 values are averages from at least three separate experiments, and replicates were typically within 25% of each other.

Clorobiocin is a more potent gyrase inhibitor than novobiocin, but only the latter compound (which was discovered first) has been introduced into clinical use (Albamycin; Upjohn/Pfizer). In the present study, we wanted to investigate the relationship between the nature of the substituents at positions 8' and 3" of novobiocin and clorobiocin and the inhibitory activity against gyrase and topo IV. For this purpose, we generated a series of compounds representing the nine permutations of H, CH3, or Cl as substituents at position 8' and H, carbamoyl, and MePC at position 3" (Fig. 1).

The total synthesis of aminocoumarins is possible but represents a time-consuming multistep procedure (9, 17). However, the biosynthetic gene clusters of novobiocin and clorobiocin have been cloned and sequenced from their respective producers, and the functions of most genes contained therein have been elucidated (12). This information lends itself to the manipulation of these pathways to produce novel structural analogs of the aminocoumarin antibiotics (13). Therefore, the compounds required for the present study were generated by genetic engineering of the producer strains. Additional clorobiocin derivatives, lacking the methyl group at 4"-OH of noviose and with different positions of the MePC group (Fig. 2), were also generated and examined in a similar way. This is, to our knowledge, the first report on a systematic evaluation of a series of aminocoumarin antibiotics against both gyrase and topo IV.


Figure 2
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  		FIG. 2. Series 2 of clorobiocin analogs and their inhibitory effects on DNA gyrase and topo IV. The concentrations of novclobiocins that cause 50% inhibition of gyrase supercoiling and topo IV decatenation are given. IC50 values are averages from at least three separate experiments, and replicates were typically within 25% of each other.


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MATERIALS AND METHODS
 
Protein, DNA, and drugs. GyrA and GyrB from E. coli were purified to homogeneity as described previously (15). Topo IV from E. coli was purified by a method based on that of Peng and Marians (18). Supercoiled and relaxed pBR322 DNAs, as well as topo IV enzyme buffers, were kindly provided by A. J. Howells (John Innes Enterprises). Kinetoplast DNA was from TopoGEN. The novclobiocin compounds were dissolved in dimethyl sulfoxide (DMSO).

Generation and isolation of novobiocin and clorobiocin analogs. Novclobiocin 103 was isolated from a clo-hal-defective mutant harboring the novO expression plasmid pTLO5. Transformants of Streptomyces roseochromogenes subsp. oscitans DS 12.976 (clo-hal mutant), harboring pTLO5, were generated and cultured for the production of clorobiocin derivatives as described previously (4). For preparative isolation, a total of 500 ml bacterial culture was pooled, acidified with HCl to pH 3, and extracted twice with an equal volume of ethyl acetate. The residue of the ethyl acetate extract after evaporation of the solvent was dissolved in 2 ml methanol (MeOH), passed through a glass column (2.8 cm by 100 cm) filled with 120 g Sephadex LH 20 (Amersham Biosciences, Freiburg, Germany), and eluted with methanol. The fractions after separation on Sephadex LH 20 were analyzed by high-performance liquid chromatography. Fractions containing novclobiocin 103 were pooled and further purified by high-performance liquid chromatography on a Multosphere RP18-5 column (250 by 4 mm; 5-µm particle size; C+S Chromatographie Service, Düren, Germany) at a flow rate of 1 ml/min, using a linear gradient from 40 to 100% of solvent B in 25 min (solvent A, MeOH-H2O-HCOOH [50:49:1]; solvent B, MeOH-HCOOH [99:1]) with detection at 340 nm. The purified compound was subjected to 1H nuclear magnetic resonance (1H NMR) and mass spectrometry analysis.

1H NMR spectra were measured on an AMX 400 spectrometer (Bruker, Karlsruhe, Germany) using CD3OD as the solvent. 1H NMR data were as follows: {delta}ppm 1.10 (s, 3H-6"), 1.32 (s, 3H-7"), 1.74 (br [broad] s, 6H-10, 11), 2.27 (s, 3H-8'-methyl), 3.34 (d, J = 7.2 Hz, 2H-7), 3.39 (d, J = 9.5 Hz, H-4"), 4.07 (br t, J = 3.0 Hz, H-2"), 4.15 (dd, J1 = 9.5 Hz, J2 = 3.0 Hz, H-3"), 5.35 (br t, J = 7.2, H-8), 5.56 (br s, H-1"), 6.83 (d, J = 8.2 Hz, H-5), 7.19 (d, J = 9.0 Hz, H-6'), 7.72 (br d, J = 8.2 Hz, H-6), 7.76 (br s, H-2), 7.81 (d, J = 9.0 Hz, H-5').

Negative fast atom bombardment mass spectra were recorded on a TSQ70 spectrometer (Finnigan, Bremen, Germany) using diethylethanolamine as the matrix. m/z (relative intensity expressed as a percentage): 568 (65, [M-H]–), 416 (13), 394 (17), 380 (30), 321 (23), 297 (16), 257 (62), 209 (100).

Novclobiocins 101, 103, 104, 105, 107, 112, 113, 114, 117, and 120 were produced as described previously (3-5, 24). A summary of the lineage of the novclobiocins is given in Fig. 3.


Figure 3
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  		FIG. 3. Lineage of novclobiocins. The scheme shows the routes for the generation of novobiocin and clorobiocin analogs by metabolic engineering of the producer strains. Series 1 is above the line; series 2 is below.

Enzyme assays. Gyrase supercoiling assays were carried out as described previously (7, 19). Samples (30 µl) contained enzyme (3.6 nM GyrA dimer, 6.4 nM GyrB dimer), 0.5 µg relaxed pBR322, a range of novclobiocin concentrations, and 1% DMSO (to test inhibition at 400 to 500 µM concentrations, this was increased to 10% in the novclobiocin titrations) and were incubated for 30 min at 37°C in the presence of 1.3 mM ATP. The reactions were carried out at 37°C and terminated after 30 min by the addition of an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1). The DNA was prepared for electrophoresis by brief vortexing, centrifugation for 5 min (15,700 x g), and addition of 15 µl of 40% sucrose-0.1 M Tris · HCl (pH 7.5)-0.1 M EDTA-bromophenol blue to the upper phase. The products were analyzed on 0.8% agarose gels; gyrase activity was estimated from the intensity of the supercoiled DNA band.

Topo IV decatenation assays were carried out by incubating enzyme (0.3 nM ParC dimer, 0.5 nM ParE dimer) and 200 ng of kinetoplast DNA with various concentrations of the novclobiocins in 40 mM HEPES · KOH (pH 7.6), 100 mM potassium glutamate, 10 mM magnesium acetate, 10 mM dithiothreitol, 2 µg/ml tRNA, 1 mM ATP, 50 µg/ml albumin, and 2% DMSO in a 30-µl volume. The reactions were carried out at 37°C and terminated after 30 min as described above. The DNA was prepared for electrophoresis as described above, and the products were analyzed on 1% agarose gels. Topo IV activity was estimated from the intensity of the minicircle band.


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RESULTS AND DISCUSSION
 
Generation of novobiocin and clorobiocin analogs. As shown in Fig. 1, the structure-activity investigations in the present study required a systematic variation of the substituents of aminocoumarin antibiotics at position 8' of the coumarin ring and at the 3"-OH group of noviose. These compounds were generated by genetic methods (Fig. 3). Novobiocin can be conveniently produced in the heterologous host Streptomyces coelicolor M512 after stable integration of the complete novobiocin biosynthetic gene cluster into the genome of this host (2). The productivity of the heterologous producer (31 mg/liter) is similar to that of the natural producer, Streptomyces spheroides (35 mg/liter), but the heterologous producer is far more amenable to genetic manipulation. Inactivation of the methyltransferase gene novO, responsible for the 8'-methylation of the aminocoumarin ring (3), within the novobiocin cluster readily yielded a mutant that accumulated novclobiocin 117 (30 mg/liter), the novobiocin analog lacking the 8'-methyl group.

When the halogenase gene clo-hal (4), responsible for the 8'-halogenation of the aminocoumarin ring in clorobiocin biosythesis, was expressed in the novO-defective strain, the hybrid antibiotic novclobiocin 114, which carries the 3"-O-carbamoyl group typical of novobiocin and the 8'-chlorine typical of clorobiocin (yield, 14 mg/liter), was produced.

The clorobiocin producer S. roseochromogenes can be manipulated genetically. We inactivated the halogenase gene clo-hal in this strain, which resulted in the production of the 8'-unsubstituted compound novclobiocin 101 (40 mg/liter). Subsequently, the methyltransferase gene novO was expressed in this strain. This led to the formation of the hybrid antibiotic novclobiocin 102, which carries the 3"-O-MePC group typical of clorobiocin and the 8'-methyl typical of novobiocin (yield, 43 mg/liter). Besides novclobiocin 102, a side product was isolated and identified as novclobiocin 103 (11 mg/liter), carrying the methyl group at 8' but no substituent at 3"-OH. This was a welcome finding, since it made a separate experiment for the generation of novclobiocin 103, by inactivation of novN in the novobiocin cluster, unnecessary.

The attachment of the MePC moiety to the 3"-OH of noviose requires, besides other proteins, the acyltransferase CloN2 (24). When we inactivated the cloN2 gene in the clorobiocin producer S. roseochromogenes, the resulting mutant accumulated, as expected, novclobiocin 104, lacking the MePC unit. This compound was produced at a yield of 85 mg/liter, surpassing the productivity of clorobiocin in the wild type. In addition, 73 mg/liter novclobiocin 105 was produced; this compound also lacks the methyl group at 4"-OH (see below, series 2). This indicates that, at high productivity, methylation at 4"-OH may become a limiting step in antibiotic production in this strain. The cloN2 gene was also inactivated in the clo-hal-defective mutant described above. As expected, the resulting clo-hal cloN2 double mutant produced novclobiocin 107, carrying no substituent at 8' and at 3"-OH. Therefore, all desired compounds of this series (Fig. 1) could be obtained readily from genetically engineered producer strains.

In an additional experiment, we intended to remove the methyl group attached to 4"-OH of the noviose moiety of clorobiocin. Inactivation of the respective O-methyltransferase gene, cloP, in a heterologous clorobiocin producer (5) readily gave the desired compound, novclobiocin 112 (Fig. 2), at a yield of 13 mg/liter. This product was accompanied by two structural isomers, novclobiocins 113 and 120, produced at yields of 6 and 4 mg/liter, respectively. These two compounds carried the MePC moiety at 2"-OH and 4"-OH of noviose, respectively, rather than at 3"-OH as in clorobiocin (Fig. 2). A further clorobiocin analog, novclobiocin 105, which lacks both the MePC moiety and the 4"-OH methyl group, had been isolated from the cloN2-defective mutant as described above.

Inhibition of DNA gyrase by novobiocin and clorobiocin analogs (series 1). The first series of novclobiocins, containing different substitutions at 3"-OH of noviose and 8' of the aminocoumarin (Fig. 1), was tested for inhibitory activity toward DNA gyrase. As exemplified in Fig. 4, the compounds were titrated into a gyrase supercoiling assay and a 50% inhibitory concentration (IC50) was estimated; novobiocin and clorobiocin were used as control compounds for comparison. As shown in Fig. 1, the presence of an acyl moiety at the 3"-OH group of noviose was very important for inhibitory activity: of the three compounds lacking such an acyl group, novclobiocin 104 (8'-Cl) showed only weak activity and novclobiocins 103 (8'-CH3) and 107 (8'-H) were inactive in the concentration range tested.


Figure 4
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  		FIG. 4. Typical data for determination of IC50 for gyrase-catalyzed supercoiling. The upper panel is an agarose gel showing the inhibition of supercoiling by novclobiocin 101. The lower panel shows the quantitation of these data and determination of the IC50. NC, nicked-circular plasmid; R, relaxed; SC, supercoiled.

Clearly, the compounds carrying an MePC moiety at 3" were more powerful as gyrase inhibitors than the compounds with a carbamoyl group. While the MePC moiety interacts directly with gyrase, the interaction of the carbamoyl group is mediated by ordered water molecules (23). Apparently, this indirect interaction results in weaker binding, and additional hydrophobic interactions via an 8'-CH3 or an 8'-Cl substituent are required for effective gyrase inhibition. In contrast, if an MePC moiety is attached to the 3"-OH group, inhibition is observed even in the absence of an 8' substituent. However, the presence of either CH3 or Cl at position 8' still enhances the activity.

Interestingly, the relative activities of CH3 and Cl depended on the nature of the acyl group at 3": in the presence of a carbamoyl group at 3", CH3 resulted in higher activity. In contrast, in the presence of a 3"-O-MePC group, Cl resulted in higher activity. Therefore, interaction with the MePC moiety seems either to enhance an interaction of the chlorine atom with the protein or to reduce a hydrophobic contact of the methyl group with the protein. This finding suggests that the structures of both natural compounds, novobiocin and clorobiocin, are highly evolved for optimal interaction with their target, DNA gyrase.

Inhibition of topo IV by novobiocin and clorobiocin analogs (series 1). The first series of novclobiocins was also titrated into topo IV decatenation assays in order to estimate IC50s toward this enzyme; novobiocin and clorobiocin were again used as control compounds. The assay is exemplified in Fig. 5. The highest concentrations tested in the topo IV assays were lower than those in the assays for gyrase due to the increased sensitivity of topo IV to DMSO, which was used as the solvent for the antibiotic. As shown in Fig. 1, inhibition of topo IV required higher concentrations of the aminocoumarins than inhibition of gyrase, a finding in accordance with earlier data (18). The relative activities of the different compounds toward topo IV showed a pattern very similar to that of the relative gyrase-inhibitory activities. However, the very effective inhibition of topo IV by clorobiocin was remarkable. Apparently, this antibiotic has evolved for optimal interaction with its targets, gyrase and topo IV. In this context, it is noteworthy that the clorobiocin gene cluster contains two resistance genes protecting the natural clorobiocin producer Streptomyces roseochromogenes from the toxic effect of the antibiotic: gyrBR, which encodes an aminocoumarin-resistant gyrase subunit, and parYR, which encodes an aminocoumarin-resistant topo IV subunit (20). In contrast, the novobiocin biosynthetic gene cluster contains only a gyrBR resistance gene (21, 22). Apparently, the weak activity of novobiocin against topo IV (Fig. 1) makes it unnecessary for the producer strain to protect its own topo IV. The biosynthetic gene clusters of novobiocin and clorobiocin have, therefore, evolved not only for the production of optimal antibiotics but also to provide, in each case, the appropriate self-protection to the producer strain.


Figure 5
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  		FIG. 5. Typical data for determination of IC50 for topo IV-catalyzed decatenation. The upper panel is an agarose gel showing the inhibition of decatenation by novclobiocin 113. The lower panel shows the quantitation of these data and determination of the IC50.

Inhibition of DNA gyrase and topo IV by clorobiocin analogs with different substitution patterns of noviose (series 2). Clorobiocin was clearly the most active compound of all aminocoumarins tested in series 1 (Fig. 1). In order to investigate further structural modifications of clorobiocin, a second series of compounds, clorobiocin analogs that contained different substitutions at the hydroxyl groups of the noviose sugar moiety (Fig. 2), were tested for their inhibition of gyrase-mediated supercoiling and topo IV-mediated decatenation; clorobiocin results are shown for comparison. Removal of the methyl group, resulting in novclobiocin 112, led to a pronounced decrease in activity (25-fold for gyrase inhibition, 50-fold for topo IV inhibition). Apparently, the hydrophobic interactions of this methyl group with the target proteins are quite important, a finding that needs to be considered in future structural modifications, e.g., chemical modifications of the 4"-OH of novclobiocin 112 by different alkylation or acylation reactions.

Three different positions of the MePC moiety within the antibiotic were investigated (Fig. 2). The canonical position in clorobiocin, 3" (i.e., novclobiocin 112), clearly resulted in the highest activity, with 2" (novclobiocin 113) and especially 4" (novclobiocin 120) resulting in less active compounds. As expected, complete removal of both the MePC and the 4"-methyl group resulted in an inactive substance.

Antibacterial activities of novclobiocins. The main emphasis of this paper is the structure-activity relationships of novclobiocins with their potential targets, DNA gyrase and topoisomerase IV. A comprehensive evaluation of their antibacterial activities against a range of relevant bacteria is beyond the scope of this work. However, we have previously evaluated the antibacterial activities of selected novclobiocins against a variety of microorganisms, including Bacillus subtilis and a range of pathogens (6). For comparison, we give the relative activities of the novclobiocins described in this paper against B. subtilis in Table 1. The compounds in Fig. 1 and 2 show, in general, similar relative activities in vivo against bacteria as they do in vitro against their enzyme targets. However, there are exceptions, most notably the higher potency of novobiocin against B. subtilis compared to clorobiocin. The same observation has been made previously, and it is at variance with the susceptibilities of other bacteria to the two antibiotics (6).


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  		TABLE 1. Activities of novclobiocins in disk diffusion assays against B. subtilis ATCC 14893

Conclusions. In summary, our experiments suggest that clorobiocin, which has not yet been tested clinically, may be a more promising lead for drug development than novobiocin. Clorobiocin shows not only 10-fold-higher gyrase-inhibitory activity than novobiocin but also 70-fold-higher topo IV inhibition. The biological significance of the topo IV inhibition is underlined by the fact that the clorobiocin producer strain, but not the novobiocin producer strain, contains a gene for an aminocoumarin-resistant topo IV subunit. Since clorobiocin attacks two distinct targets, gyrase and topo IV, development of resistance against clorobiocin is expected to proceed less readily than development of resistance against novobiocin, which has been a limiting factor in the clinical use of the latter antibiotic. The structure of clorobiocin appears to be highly evolved for an optimal interaction with its targets. If structural modifications are attempted, our study suggests that the compounds should still contain a 3" acyl group and an 8' substituent. Variations may be explored, e.g., for the nature of the acyl group at 3", the hydrophobic substitutent at 4"-O, and the acyl group attached to the 3-amino group of the aminocoumarin core.

Our results demonstrate that we can systematically alter drug structures by metabolic engineering of the producer strains and assess the effects of the resulting compounds on drug targets. This provides a useful knowledge base for developing new drugs and further derivatives with improved properties. It further supports the use of recombinant techniques in producing novel antibiotics by exploiting natural systems, avoiding the need for time-consuming multistep organic synthesis.


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ACKNOWLEDGMENTS
 
We thank John Innes Enterprises for supplying relaxed pBR322 and topo IV enzyme buffers and Melisa Wall for help with making topo IV. We thank Hui Xu and Heike Rapp for isolation of various novclobiocins.

This work was supported by a grant from the European Commission (Combigyrase LSHB-CT-2004-503466).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, United Kingdom. Phone: 44 1603 450771. Fax: 44 1603 450018. E-mail: tony.maxwell{at}bbsrc.ac.uk. Back


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Antimicrobial Agents and Chemotherapy, April 2006, p. 1136-1142, Vol. 50, No. 4
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.4.1136-1142.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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