Actinomycetes are known for their large genetic potential to produce several distinct classes of secondary metabolites.While cultivating Actinomadura sp. DSMZ 13491, the producer of the cyclic heptapeptide GE23077 it was observed to produce an antibacterial activity.Despite being a potent and selective inhibitor of bacterial RNA polymerase, GE23077 is devoid of activity against bacterial cells, except for marginal activity against some Moraxella spp.Here, the isolation, structure elucidation and properties of madurastatin C1 (1), the antimicrobial compound produced by this actinomycete, are reported.A 10-mL seed culture of Actinomadura sp. DSMZ 13491, prepared by inoculating a frozen cell stock into 30 mL of AFT medium (2% glucose, 0.2% yeast extract, 0.8% soybean meal, 0.5% tryptone, 0.1% NaCl, 0.4% CaCO3, adjusted to pH 7.2 with 0.1 N NaOH before sterilization) and incubating for 72-96 h, was transferred into 200 mL of fresh AFT medium.The production of antibacterial activity was monitored over time by depositing 10 μL of a microbial extract onto Muller Hinton Agar (Difco Laboratories, Detroit, MI, USA) plates, previously inoculated with 105 cfu mL-1 Staphylococcus aureus ATCC 6538P or Micrococcus luteus ATCC 10240.An inhibition halo was clearly observed on M. luteus plates after 24 h of cultivation, whereas only a weak activity was visible on the S. aureus plates.In an HPLC anal. of the culture extract, a major peak was observed at 2.6 min with a m/z of 592 [M+H]+, whereas the two major congeners of GE23077, with m/z of 804 and 806, were observed at 1.8 min, as expected.Minor peaks were also observed at 2.1 min, with m/z of 610 and 645.The 2.6-min metabolite was purified by medium pressure chromatog. on a reverse-phase C18 RediSep RF column, using a CombiFlash RF Teledyne Isco Medium Pressure Chromatog. System (Teledyne ISCO, Lincoln, NE, USA).The column was previously conditioned with a mixture of phase A (50 mM HCOONH4), phase B (CH3CN) 95:5 (volume/volume), and eluted with a 15-min linear gradient from 5 to 95% phase B.The active fractions were collected, concentrated under vacuum and lyophilized, yielding 13 mg of purified 1 (estimated at least 95% pure by NMR).NMR and MS analyses of 1 indicated high similarity with the siderophores named madurastatins.1H- and 13C-NMR data indicated the presence of an ortho disubstituted benzene, as confirmed by bidimensional experiments showing four aromatic protons with a TOCSY correlation.In particular, a doublet at 7.61 ppm had a COSY correlation with a pseudo-triplet at 6.82 ppm, and a doublet at 6.89 ppm correlated with a pseudo-triplet at 7.34 ppm, whereas the pseudo-triplet signals intercorrelated.HMBC experiments demonstrated the disubstituted benzene to be a salicylic acid residue.Five aliphatic spin systems were also observed by using homo- and hetero-nuclear 2D-NMR experimentsCOSY and TOCSY correlations evidenced the presence of an aziridine group and of a six-membered lactam at the C-terminus.A strong HMBC correlation between the Me group at 2.54 ppm and the α-carbon at 61.3 ppm confirmed the presence of an N-Me group.NMR data demonstrated that 1 is a pentapeptide carrying a salicylate at the N-terminus.Thus, 1 is highly related to madurastatin A1, except for the lack of the C-16 Me, which probably originates from incorporation of glycine instead of alanine.This is confirmed by the occurrence of a signal at 3.82 ppm, integrating two protons, corresponding to the CH2 at position 16.The structure of 1 as established by NMR is also supported by MSn anal.Its fragmentation pathway showed a typical peptide fragmentation, identical to that reported for madurastatin A1, except for the difference at C-16, whereas 1 produced fragments of m/z 219 and 403, ions of m/z 233 and 417 were reported for madurastatin A1.As described for madurastatin A1, a characteristic bathochromic shift (from 254 and 310 nm to 280 and 445 nm) was observed when 1 mg was added to 1 mL 0.6 M FeCl3, confirming the high chelating affinity for ferric ions.By analogy with madurastatin A1, it can be inferred that m/z 610 and 645 present in the 2.1-min peak correspond to 1 hydrolyzed at the aziridine ring (position C-19) and to the 1-Fe+3 complex, resp.Consistently, the compound at m/z 610 yields an MS fragment at 480 amu, instead of 462 amu as seen with 1.Moreover, the conversion of the m/z 592 to 645 was observed upon addition of 1 to the FeCl3 aqueous solutionAlthough inhibition halos of 25 and 5 mm were observed with M. luteus and S. aureus, resp., when spotting 50 μg of 1 onto Muller Hinton Agar plates, neither 1-hydrolyzed nor 1-Fe3+ complex showed antimicrobial activity when tested with M. luteus, confirming the important role of the aziridine ring for the biol. activity.In conclusion, Actinomadura sp. DSMZ 13491 can produce a second bioactive metabolite in addition to GE23077.Earlier experiments had indeed shown that the genome of this strain encodes at least 12 nonribosomal peptide modules.Although the relatedness between the Actinomadura strain described by Harada et al. and the present one is not known, it is remarkable that a sputum- and a soil-derived strain produce very similar compounds without the need for iron limited media, as seen instead with other actinomycetes.
