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Pharmazeutische Biologie, Pharmazeutisches Institut, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany
Correspondence
Lutz Heide
heide{at}uni-tuebingen.de
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). All three compounds contain an unusual branched deoxysugar with a 5,5-gem-dimethyl structure (Piepersberg & Distler, 1997). In novobiocin, the 3-OH group of this sugar is acylated with a carbamoyl group, resulting in noviose. In clorobiocin and coumermycin A1, the 3-OH group is acylated with a 5-methyl pyrrole-2-carboxyl moiety (Fig. 1). Feeding experiments with isotope-labelled precursors showed that one of the methyl groups at C-5 of noviose is derived from glucose (Birch et al., 1962), the other from S-adenosylmethionine (SAM) (Birch et al., 1960). Cloning of the biosynthetic gene clusters of novobiocin, clorobiocin and coumermycin A1 (Pojer et al., 2002; Steffensky et al., 2000; Wang et al., 2000) revealed a contiguous group of five genes, novSTUVW and the homologous cloSTUVW and couSTUVW, which are contained in all three clusters in identical order. Sequence analysis suggested that these genes are responsible for the biosynthesis of the deoxysugar moieties of the antibiotics: NovV shows sequence similarity to dTDP-glucose synthases, NovT to dTPD-glucose-4,6-dehydratases, NovW to dTPD-4-keto-6-deoxyglucose epimerases and NovS to dTPD-4-keto-hexose reductases. Such enzymes are commonly found in deoxysugar pathways (He & Liu, 2002).
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). It has therefore been speculated that NovU catalyses C-5 methylation in the biosynthesis of noviose, and CloU and CouU the corresponding reactions in clorobiocin and coumermycin A1 biosynthesis (Li & Heide, 2004).
NovW, and its homologues CloW and CouW, show sequence similarity to (d)NDP-4-keto-6-deoxyglucose epimerases. These include, for example, the di-epimerase RmlC which carries out an epimerization of both C-3 and C-5 in the biosynthesis of L-rhamnose in E. coli (Giraud et al., 2000) and the mono-epimerase WbcA which carries out an epimerization of only C-3 in the biosynthesis of 6-deoxy-D-gulose during the formation of the O-antigen of Yersinia enterocolitica (Trefzer et al., 1999). L-rhamnose and 6-deoxy-L-gulose differ only in the stereochemistry at C-5.
From chemical reasoning, three pathways appear equally plausible for the formation of noviose from dTPD-4-keto-6-deoxyglucose: (a) NovW may catalyse a 3,5-epimerization, followed by a C-5 methylation by NovU and a 4-keto reduction by NovS (this pathway is depicted in Fig. 1
); (b) NovW may catalyse only a 3-epimerization, followed by a C-5 methylation by NovU (in opposite stereochemistry to above) and a 4-keto reduction by NovS; (c) NovU may methylate C-5 of dTPD-4-keto-6-deoxyglucose, followed by a 3-epimerization by NovW and a 4-keto reduction by NovS. All three reaction sequences would lead to the same activated deoxysugar, which is known to be transferred to the aglycon under catalysis of the glycosyltransferase NovM (Albermann et al., 2003; Freel Meyers et al., 2003). 4-O-Methylation and 3-O-acylation have been shown to occur after the glycosyl transfer (Li & Heide, 2004).
In a first biochemical analysis of NovWUS, Thuy et al. (2005) incubated dTPD-4-keto-6-deoxyglucose with NovU in the presence or absence of NovW. Only in the presence of NovW did LC-MS analysis show a product with the expected mass of a methylated dTDP-4-keto-6-deoxyhexose, suggesting that epimerization by NovW must precede methylation by NovU. However, no structural elucidation of the products could be provided, and the analytical method used for investigation of the NovW product (i.e. HPLC of the dTDP-deoxysugar, either directly or after decomposition to maltol) does not allow conclusions about the stereochemistry at C-5 (Sohng et al., 2004).
Recently, the crystal structure of NovW was reported (Jakimowicz et al., 2006). It was observed that the structure of the active centre of NovW was different from that of the di-epimerase RmlC, but resembled that of the mono-epimerase EvaD. Therefore, Jakimowicz et al. (2006) raised the question whether NovW might function as a 3-mono-epimerase rather than as a 3,5-di-epimerase.
We decided to carry out an in-frame inactivation of the cloU gene in the clorobiocin biosynthetic gene cluster. NMR investigation of a (potentially) resulting clorobiocin derivative lacking one of the methyl groups at C-5 of the deoxysugar would allow unequivocal determination of whether the sugar moiety is an L-rhamnose derivative or a 6-deoxy-D-gulose derivative, and would therefore clarify whether deoxysugar biosynthesis proceeds via 3,5-epimerization or via 3-epimerization. At the same time, such an experiment would present the first example of metabolic engineering of the deoxysugar biosynthetic pathway of the aminocoumarin antibiotics. This antibiotic class has been very successfully used in the past for the generation of new compounds, using metabolic engineering of the biosynthetic pathways of the aglycon of the antibiotic as well as engineering of the tailoring reactions (Li & Heide, 2005). Finally, identification of the function and the substrate of cloU would make this unusual 5-C-methyltransferase gene available for use in the metabolic engineering of deoxysugar pathways, as described by Salas and coworkers (Lombo et al., 2004; Perez et al., 2005; Rodriguez et al., 2002).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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redD actII-ORF4 SCP1– SCP2–) (Floriano & Bibb, 1996) was cultured as described previously (Eustáquio et al., 2004; Kieser et al., 2000). Escherichia coli strains ET12567 (MacNeil et al., 1992) and XL-1 Blue MRF' (Stratagene) were used for cloning experiments and grown as described by Sambrook & Russell (2001). The REDIRECT technology kit for PCR targeting (Gust et al., 2003) was obtained from Plant Bioscience Limited. Kanamycin (50 µg ml–1), chloramphenicol (25–50 µg ml–1), apramycin (50 µg ml–1) and thiostrepton (25 µg ml–1) were used for selection of recombinant strains. Cosmid clo-BG1 and plasmid pUG019 have been described previously (Eustáquio et al., 2005a).
Genetic procedures.
Standard procedures for DNA isolation and manipulation were performed as described by Kieser et al. (2000) and Sambrook & Russell (2001). Isolation of cosmids and plasmids was carried out with ion-exchange columns (Nucleobond AX kits; Macherey–Nagel), according to the manufacturer's protocol. Genomic DNA was isolated from Streptomyces strains by lysozyme treatment and phenol/chloroform extraction (Kieser et al., 2000). Southern blot analysis was performed on Hybond-N nylon membrane (Amersham Biosciences) with digoxigenin-labelled probe using the DIG high prime DNA labelling and detection starter kit II (Roche Applied Science).
Replacement of cloU with aac(3)IV and construction of cosmids clo-AF1/clo-AF2.
In cosmid clo-BG1, cloU was replaced, via 
-Red-mediated recombination (Eustáquio et al., 2005a), by an apramycin resistance cassette, aac(3)IV. The resistance cassette was excised from pUG019 (Eustáquio et al., 2005a) by digestion with EcoRI and HindIII, and was generated by PCR with the primer pair cloU-f (5'-GACCGGCGGTTTGCATCCCGTGAGGGGGAGGAAGAGTTGATTCCGGGGATCTCTAGATC-3') and cloU-r (5'-TGGAACGGGATCACCGCAGCCGACACCGTGGCCACGTCAACTAGTCTGGAGCTGCTTC-3'). Bold letters represent 39-nt homologous extensions to the DNA regions immediately upstream and downstream of cloU, including the putative start and stop codons of cloU. Underlined letters indicate XbaI and SpeI restriction sites.
PCR was carried out in a 50 µl volume with 50 ng template, 0.2 mM dNTPs, 50 pmol each primer and 5 % (v/v) DMSO, using the Expand High Fidelity PCR system (Roche Molecular Biochemicals): denaturation at 94 °C for 2 min, then 10 cycles of denaturation at 94 °C for 45 s, annealing at 45 °C for 45 s and extension at 72 °C for 90 s, followed by 20 cycles of annealing at 48 °C, and the last elongation step at 72 °C for 5 min. The PCR product was introduced by electroporation into E. coli BW25113(pIJ790) harbouring cosmid clo-BG1 (Gust et al., 2003). The modified cosmid was isolated and analysed by restriction enzyme digestion. This cosmid was termed clo-AF1. For the excision of the apramycin resistance cassette from clo-AF1, cosmid DNA was isolated from E. coli ET12567, digested with XbaI and SpeI and religated overnight at 16 °C to give cosmid clo-AF2.
Heterologous expression of clo-AF2 in S. coelicolor M512.
Because of the potent methylation restriction system of S. coelicolor, cosmid DNA had to be passed through a non-methylating host. We used E. coli ET12567 for this purpose (Eustáquio et al., 2005a). The modified cosmid clo-AF2, still carrying the kanamycin resistance gene aphII, was then introduced into S. coelicolor M512 via PEG-mediated protoplast transformation (Kieser et al., 2000). Clones resistant to kanamycin were selected and checked for site-specific integration into the genome by Southern blot analysis.
Complementation of the cloU mutant with the cloU gene.
For the complementation assays with cloU, vector pAF16 was constructed. A fragment of 1353 bp containing the whole sequence of cloU [position 36566–37919 in sequence AF329398 (GenBank accession no.)] was obtained by PCR amplification with primer pair k-cloU/BamHI (5'-GACCGGCGGTTTGGATCCCGTGA-3') and k-cloU/XbaI (5'-TTCCGACCTTCTAGACACGTGG-3') using cosmid clo-BG1 as template. The underlined letters represent mutations inserted into the original sequence to give the desired BamHI and XbaI restriction sites, respectively. The purified PCR product was restricted with BamHI and XbaI and ligated into the same sites of pBluescript SK(–) (Stratagene) to give pAF15. The BamHI–XbaI fragment of 1.3 kb from pAF15 was religated into the BamHI and XbaI restriction sites of the replicative vector pUWL201 (Doumith et al., 2000), downstream of the constitutive ermE* promoter, to give pAF16.
After passing through the non-methylating strain E. coli ET12567, pAF16 was introduced into the cloU-defective mutant using PEG-mediated protoplast transformation (Kieser et al., 2000). After transformation, thiostrepton-resistant clones were selected. To confirm the presence of intact pAF16, the plasmid was reisolated by alkaline lysis and potassium acetate precipitation, procedure D (Kieser et al., 2000). Subsequently, plasmid DNA isolated from Streptomyces was amplified in E. coli XL-1 Blue MRF' before restriction analysis.
Analysis of secondary metabolites.
Transformants of S. coelicolor, harbouring clo-BG1, clo-AF2 and clo-AF2 with pAF16, respectively, were cultured and assayed for the production of clorobiocin derivatives by HPLC as described previously (Eustáquio et al., 2004) using a Multosphere RP18-5 column (250x4 mm; 5 µm; CS Chromatographie Service, Düren, Germany) at a flow rate of 1 ml min–1, using a linear gradient from 60 to 100 % solvent B over 30 min (solvent A, MeOH/H2O/HCOOH 20 : 79 : 1; solvent B, MeOH/HCOOH 99 : 1) with detection at 340 nm. Authentic clorobiocin (Aventis) was used as standard.
For preparative isolation, cultivation was carried out in 500 ml baffled flasks containing 50 ml production medium as described above. A total of 5 l bacterial cultures of the cloU-defective strain was pooled, acidified with HCl to pH 4 and extracted with ethyl acetate after prior removal of the lipophilic components by extraction with petroleum ether. The residue of the ethyl acetate extract after evaporation of the solvent was dissolved in 30 ml methanol and further purified on a semipreparative HPLC column (Multosphere 120 RP18-5, 5 µm, 250x10 mm; CS Chromatographie Service, Düren, Germany) using the same solvents and gradient as for the analytical column, but with a flow rate of 2.5 ml min–1. The purified compounds were subjected to 1H-NMR and MS analysis.
Negative-ion FAB mass spectra were recorded on a TSQ70 spectrometer (Finnigan) using diethanolamine as matrix.
The negative ion fast atom bombardment mass spectrum of novclobiocic acid 106 showed molar ion at m/z=380 ([M-H]–). Clorobiocic acid gave the following negative ions: m/z=414 ([M-H]–) and 225. Novclobiocin 122 and novclobiocin 123 showed the molar ion at m/z=681 ([M-H]–).
1H-NMR spectra were acquired on an AC 250 spectrometer (Bruker) or an AMX 400 spectrometer (Bruker). The NMR data for clorobiocin, novclobiocic acid 106, clorobiocic acid, novclobiocin 122 and 123 are shown in Table 1.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and 88 % to NovU from Streptomyces spheroides (Steffensky et al., 2000). CouU and NovU were putatively assigned to the C-methyltransferase reactions involved in the deoxysugar biosynthesis of coumermycin A1 and novobiocin (Li & Heide, 2004). CloU contains a possible SAM-binding site (Kagan & Clarke, 1994), located at position 108–116 in CloU (VVEFGSNTG). NovU and CouU contain the same motif at the same position.
Inactivation of cloU
For inactivation of cloU, the gene was replaced by an apramycin resistance gene, aac(3)IV, in cosmid clo-BG1 containing the complete biosynthetic gene cluster of clorobiocin. The resistance cassette was then removed and the modified cosmid was expressed in a heterologous producer strain as described below.
Cosmid clo-BG1, containing the clorobiocin gene cluster, the attachment site attP, the integrase gene of bacteriophage 
C31, and tetracycline and neomycin resistance genes, had been constructed in a previous study (Eustáquio et al., 2005a). Site-specific integration of this cosmid into the attB site of genome of S. coelicolor M512 had resulted in the formation of clorobiocin by the heterologous host, in amounts comparable to the wild-type producer strain (Eustáquio et al., 2005a).
The apramycin resistance gene, aac(3)IV, was amplified from plasmid pUG019 (Eustáquio et al., 2005a) by PCR using primers with 39 bp extensions homologous to the regions upstream and downstream of cloU. The PCR product was used to replace the entire ORF of cloU in cosmid clo-BG1, leaving only the start and the stop codons intact. This gave the modified cosmid clo-AF1. The resistance cassette was then removed by digestion with XbaI and SpeI, enzymes which create compatible ends, allowing religation of the outer ends and subsequent excision of the cassette. The removal of the acc(3)IV cassette was carried out to avoid possible polar effects of this cassette on downstream genes. The resulting cosmid was termed clo-AF2 (Fig. 2a, c).
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). Mutants resulting from integration of this cosmid into the host genome were selected by their kanamycin resistance. Five independent mutants were examined by Southern blot analysis, showing identical genotypes. Results of one such mutant are presented in Fig. 3. The desired deletion of cloU in the mutant was clearly shown by the disappearance of the 1 kb BglII restriction fragment arising from the BglII site within cloU, in comparison to the clo-BG1 strain (Fig. 2b, d).
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; Pojer et al., 2002). Ethyl acetate extracts of the cultures were analysed by HPLC.
The integration mutant of S. coelicolor M512 harbouring the intact clorobiocin cluster (clo-BG1) showed clorobiocin as the dominant product, accompanied by its non-chlorinated derivative novclobiocin 101 and its structural isomer isoclorobiocin (Figs 3 and 4a). In contrast, production of clorobiocin was not detected in the cloU-defective strains. Instead, these mutants produced the aglycon of clorobiocin, clorobiocic acid (20 mg l–1) as well as the corresponding non-chlorinated compound, novclobiocic acid 106 (39 mg l–1). Both compounds were isolated on a preparative scale from 1 l culture and identified by NMR and MS analysis (see below). Besides these major products, we found two minor peaks with retention times of 20 and 23 min which were designated novclobiocin 122 and novclobiocin 123, respectively (Figs 3 and 4b).
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Structural elucidation
The mass spectrum of clorobiocic acid showed a molecular ion at m/z 414 ([M-H]–), 281 mass units less than that of clorobiocin, corresponding to the lack of the entire deoxysugar moiety. This result was confirmed by the absence of the signals of sugar protons and the signals for the protons of the 5-methylpyrrole-2-carbonyl moiety in 1H-NMR analysis, in comparison to those of clorobiocin. In contrast, the signals for the protons of the aminocoumarin and 3-dimethylallyl-4 hydroxybenzoyl unit were clearly observed (Table 1).
Novclobiocic acid 106 showed a molecular ion at m/z 380 ([M-H]–) in MS analysis, 35 mass units smaller than that of clorobiocic acid, corresponding to loss of the chlorine atom. This was confirmed by the signal of an additional proton at 6.66 p.p.m. for 8'-H. The 1H-NMR data of this compound (Table 1) corresponded well to the data obtained in a previous study (Eustáquio et al., 2005b).
The 1H-NMR analysis of novclobiocin 122 and novclobiocin 123 showed similar signals to clorobiocin for the protons of the aminocoumarin moiety, the prenylated 4-hydroxybenzoyl moiety and the 5-methylpyrrole-2-carbonyl moiety. However, the signals of the deoxysugar moiety were clearly different in comparison to those of clorobiocin. The deoxysugar moiety in all three compounds is present in the 1C4 conformation. The protons at 1'' and 2'' are thus in equatorial positions, whereas those at 3'' and 4'' are in axial positions. Accordingly, in clorobiocin, 3''-H shows strong coupling with 4''-H (J=10.3 Hz), but weaker coupling with 2''-H (J=2.8 Hz), resulting in a characteristic doublet of doublets signal for 3''-H (J=2.8 and 10.3 Hz) and a doublet for 4''-H (J=10.3 Hz). The equatorial 2''-H shows additional coupling with the equatorial 1''-H with a coupling constant of 1.8 Hz, resulting in a triplet signal for 2''-H due to low resolution of the measurement. The two methyl groups at position 5 of the deoxysugar appear as singlets at 1.18 and 1.35 p.p.m. in the NMR spectrum of clorobiocin. In contrast, in novclobiocin 122, only one methyl group was observed as a doublet at 1.18 p.p.m. with a coupling constant of 6.4 Hz. The proton at 4'' appeared as a triplet with a coupling constant of 9.6 Hz. This means that the proton at 4'' has a strong coupling both with 5''-H and 3''-H. This proves that all of these protons are located in axial positions and therefore the deoxysugar moiety is an L-rhamnose derivative. The negative mass spectra of this compound showed a molar ion peak at m/z 681 ([M-H]–), 14 mass units smaller than that of clorobiocin, corresponding to a loss of a methyl group in comparison to clorobiocin.
In novclobiocin 123, the proton at 3'' appears at 4.29 p.p.m. In contrast, the signal for 2''-H is shifted strongly downfield to 5.36 p.p.m. This shows that the acyl moiety is attached to 2''-OH rather than to 3''-OH, similar to observations in previous studies (Freitag et al., 2005; Galm et al., 2004). The negative mass spectra showed the same molar ion as observed for novclobiocin 122.
The amount of novclobiocin 122 produced by the cloU-defective strain was 1.5 mg l–1; the amount of novclobiocin 123 was 0.4 mg l–1.
Complementation of the cloU mutation
To prove that only the inactivation of cloU was responsible for the changes in secondary metabolite production, the cloU-defective strain was complemented by expression of an intact copy of cloU under the control of the constitutive ermE* promoter, using the expression vector pUWL201. HPLC analysis showed that the cloU-defective strain harbouring the cloU expression plasmid pAF16 produced clorobiocin at a similar level to the S. coelicolor M512 strain carrying the complete biosynthetic gene cluster of clorobiocin (Fig. 4c).
Antibacterial activity of the new novclobiocins in comparison to clorobiocin
The novclobiocins obtained (novclobiocin 122 and novclobiocin 123) were assayed for antibiotic activity against Bacillus subtilis ATCC 14893 in comparison to authentic clorobiocin. Novclobiocin 122 exhibited only 20 % of the activity of clorobiocin, showing that the methylation at position 5 of the deoxysugar is important for the biological activity. Transfer of the acyl moiety from 3''-OH to 2''-OH resulted in a further reduction of activity, as novclobiocin 123 was half as active as novclobiocin 122 (Fig. 5). Results from an earlier study showed that the removal of the deoxysugar moiety resulted in a 99 % activity loss (Eustáquio et al., 2004). Therefore the two acids were not analysed for their biological activity.
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DISCUSSION |
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), CloN6 (Westrich et al., 2003) and CloU (present study).
CloU and its two homologues are, to our knowledge, the first 5-C-methyltransferases identified in the biosynthesis of branched deoxysugars. Several enzymes are known to catalyse a 3-C-methylation of deoxysugars, such as TylC3 (He & Liu, 2002), EryBIII (Lombo et al., 2004) and AviG1 (Weitnauer et al., 2002). But the 5-C-methyltransferase CloU and its homologues add a unique, new function to the toolbox of deoxysugar biosynthesis genes, which can be assembled in a combinatorial fashion for the generation of a large diversity of activated deoxysugars (Lombo et al., 2004; Perez et al., 2005; Rodriguez et al., 2002; Salas & Mendez, 2005).
Inactivation of cloU led to the formation of novclobiocin 122 and 123. The deoxysugar moieties of these two clorobiocin analogues show substituted L-rhamnose structures, as clearly shown by the characteristic coupling of the deoxysugar protons. The NMR data of these compounds are in accordance with those reported for other L-rhamnosides (Igarashi et al., 2005; Ströch, 2003), and in contrast to those of 6-deoxy-D-gulosides (Patroni & Stick, 1987). This proves that in the biosynthesis of the deoxysugar moieties of the aminocoumarin antibiotics, the intermediate dTDP-4-keto-6-deoxyglucose undergoes epimerization at both C-3 and C-5. It appears most probable that the epimerase CloW catalyses both of these epimerizations, similar to the established 3,5-epimerase RmlC (Giraud et al., 2000), and in contrast to the 3-epimerase WbcA (Trefzer et al., 1999).
The recent elucidation of the structure of NovW, a close homologue of CloW, showed that a tyrosine side chain in the active centre is oriented differently than in RmlC (Jakimowicz et al., 2006), but similarly to 5-epimerase EvaD involved in chloroeremomycin biosynthesis. The genuine substrate of EvaD is a sugar which carries an amino and a methyl group at position 3; therefore, only position 5 can be epimerized easily. However, when EvaD was incubated with dTDP-4-keto-6-deoxyglucose (i.e. the genuine substrate of NovW and CloW), a rapid epimerization at C-5, but also a slow epimerization at C-3 was observed (Merkel et al., 2004). Therefore, EvaD acts as 5-monoepimerase in vivo, but can also catalyse a 3,5-double epimerization of a suitable substrate in vitro. From the structure of NovW, Jakimowicz et al. (2006) raised the question whether NovW may function as a 3-monoepimerase. Our present results, however, suggest that at least the similar CloW acts as a 3,5-double epimerase of dTDP-4-keto-6-deoxyglucose in vivo.
When the intact clorobiocin cluster was expressed in S. coelicolor M512, the major part of the aminocoumarin antibiotics was produced in form of glycosylated compounds (Fig. 4a). In contrast, after cloU inactivation, approximately 97 % of the aminocoumarins were produced in the form of aglycons and only 2.3 % in glycosylated form. This indicates that either deoxysugar biosynthesis or glycosyl transfer was impaired as a result of cloU inactivation. The possibility that cloU inactivation reduced transcription or translation of downstream genes, namely the deoxysugar biosynthesis genes cloV and cloW, could be ruled out by a complementation experiment of the cloU mutant with a cloU expression plasmid, which restored production of the glycosylated compounds to the original level. Since dTDP-L-rhamnose has been reported to be a poor substrate of the glycosyltransferase NovM (Albermann et al., 2003; Freel Meyers et al., 2003), a possible explanation is that the glycosyltransferase CloM may transfer the modified deoxysugar, which results from cloU inactivation, with less catalytic efficiency than the natural substrate.
After transfer of the deoxysugar moieties, final tailoring reactions consist of 4-O-methylation and 3-O-acylation of the deoxysugar (Li & Heide, 2004). Apparently, the substrate specificity of the enzymes involved is broad enough to allow the utilization of clorobiocin analogues lacking the methyl group at C-5 of the deoxysugar, as no metabolites were detected which lacked the substituents at 3''-OH or 4''-OH.
Novclobiocin 122 and 123 represent new clorobiocin analogues. Investigation of their antibacterial activity showed that novclobiocin 122 exhibits only 20 % of the activity of clorobiocin, indicating that the hydrophobic contacts between the 5,5-gem-dimethyl group of the deoxysugar and gyrase contribute to the binding of the antibiotic to its target (Maxwell, 1999). As observed previously (Li & Heide, 2005), shifting the 5-methylpyrrole-2-carboxyl moiety from position 3 to position 2 further reduced activity.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 16 February 2006;
revised 12 April 2006;
accepted 26 April 2006.
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is given in p.p.m.; J in Hz. Spectra were obtained at 250 or 400 MHz. The NMR spectra of clorobiocin, clorobiocic acid, novclobiocic acid 106 and novclobiocin 123 were measured in CD3OD. The NMR spectrum of novclobiocin122 was measured in CD3SOCD3. br, Broad signal; s, singlet; d, doublet; dd, double doublet; dq, double quartet; m, multiplet; t, triplet.
