Characterization of madurastatin C1, a novel siderophore from Actinomadura sp.

Abstract

Actinomycetes are known for their large genetic potential to produce several distinct classes of secondary metabolites. 1 While cultivating Actinomadura sp. DSMZ 13491, the producer of the cyclic heptapeptide GE23077, 2 we observed it produced 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. 2 Here we report on the isolation, structure elucidation and properties of madurastatin C1 (1), the antimicrobial compound produced by this actinomycete.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% CaCO 3 , 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 ml of a microbial extract, prepared as described by Pozzi et al., 3 onto Muller Hinton Agar (Difco Laboratories, Detroit, MI, USA) plates, previously inoculated with 10 5 cfu ml À1 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. An HPLC analysis of the culture extract is shown in Figure 1a. 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. 2 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 chromatography on a reverse-phase C18 RediSep RF column, using a CombiFlash RF Teledyne Isco Medium Pressure Chromatography System (Teledyne ISCO, Lincoln, NE, USA). The column was previously conditioned with a mixture of phase A (50 mM HCOONH 4 ), phase B (CH 3 CN) 95:5 (v/v), 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. 4 1 H-and 13 C-NMR data (Table 1) 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 p.p.m. had a COSY correlation with a pseudo-triplet at 6.82 p.p.m., and a doublet at 6.89 p.p.m. correlated with a pseudotriplet at 7.34 p.p.m., 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 experiments. COSY and TOCSY correlations evidenced the presence of an aziridine group...

The secondary metabolites of rare actinomycetes: chemistry and bioactivity

“…At rst, a new tetrasaccharide derivative, actinotetraose L (28), was discovered by Guo et al But the compound was not shown to display potent immunosuppressive activity and cytotoxicity from T-cell viability and MTT assays. 16 One new angucyclinone derivative, amycomycin A (29), and one new angucycline, amycomycin B (30), along with 5 known compounds, tetrangomycin (31), PD116779 (32), X-14881E (33), sakyomicin A (34) and sakyomicin C (35), also were isolated from the rare actinobacterium Amycolatopsis sp. HCa1.…”

Spoxazomicin D and Oxachelin C, Potent Neuroprotective Carboxamides from the Appalachian Coal Fire-Associated Isolate Streptomyces sp. RM-14-6

“…2–4,7,8,10,13–17,29,52–70 Likewise, including the new bacterial carboxamides 6 and 7 reported herein, over 60 benzamide derivatives have been reported from bacteria, many of which also reportedly display interesting biological and pharmaceutical properties. 71 Importantly, the current study is the first to assess spoxazomicins or oxachelins in the context of neuroprotection against EtOH-induced damage, where corresponding bioactivity comparisons among the analogues tested highlight the importance of the spoxazomicin D ( 2 ) carboxamide and oxachelin C ( 5 ) salicylate as critical to this newly discovered function.…”

“…Roughly 0.3% 1 of reported microbial metabolites are classified as phenol-oxazolines or salicylate-containing natural products, where salicylate C7 amino acid or peptide modification is a common feature (e.g., spoxazomicins, 2,3 oxachelin, 4,5 amychelin, 6 madurastatins, 7,8 acinetobactin, 9 asterobactins, 10 carboxymycobactins, 11–13 exochelins, 14 and nocardimicins 15,16 ). While most commonly reported as metal chelators/siderophores, 4,6,10 representative members have also been noted to display cancer cell line cytotoxicity and antimicrobial and antitrypanosomal activities.…”

N-Carbamoylation of 2,4-Diaminobutyrate Reroutes the Outcome in Padanamide Biosynthesis

“…For macrocyclization, a hydroxyl or an amino group of the acyl or peptidyl chain acts as an internal nucleophile to attack the carbonyl carbon of the acyl or peptidyl-O-TE and a cyclic product is released. Typically, the nucleophile is in an amino acid earlier in the peptide chain (Du and Lou, 2010), but recently, a number of siderophores have been isolated that have either a C-terminal cyclic N d -OH-ornithine (Kodani et al, 2013;Mazzei et al, 2012;Seyedsayamdost et al, 2011) or a C-terminal cyclic N ε -OH-lysine (Hoshino et al, 2011). In these cases, the hydroxylamine of the C-terminal N d -OH-ornithine or N ε -OH-lysine is thought to be used as a nucleophile to generate small, N-heterocycles and release linear peptides.…”