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Summary:
Summary The genus Rhodococcus belong to the order actinomycetes, which are gram-positive bacteria with high GC content. They produce a broad range of bioactive secondary metabolites that found use in the pharmaceutical industry and in other biotechnological applications. Most of these bioactive metabolites were derived from nonribosomal peptides (NRP) or polyketides (PK). However, only few natural products have been isolated and characterized so far. In particular, within the Rhodococcus genus, substantial chemical diversity has been observed among the iron-chelating siderophores through the structure elucidation of rhodochelin, rhodobactin and heterobactin A1. Therefore this work was focused on isolation and structural characterization of further new iron-chelating molecules to explore the possible chemical potential of this genus on secondary metabolite production. In this study we accomplished the isolation, the structural characterization and the elucidation of the biosynthetic origin of heterobactins, a catecholate-hydroxamate mixed-type siderophores from Rhodococcus erythropolis PR4. The structure elucidation of the extracted and purified siderophore heterobactin A was accomplished via MSn analysis and NMR spectroscopy and revealed the noteworthy presence of a peptide bond between the guanidine group of an arginine residue and a 2,3- dihydroxybenzoate moiety. The two other purified siderophores heterobactin S1 and S2 were found to be derivatives of heterobactin A that have sulfonation modifications on the aromatic rings. The bioinformatic analysis of the R. erythropolis PR4 genome and the subsequent genetic and biochemical characterization of the putative biosynthetic machinery identified the gene cluster responsible for the biosynthesis of the heterobactins to encode the three modules comprising nonribosomal peptide synthetase (NRPS) HtbG. Interestingly, the HtbG NRPS contains an unprecedented C-PCP-A domain organization within the second module of the HtbG-synthetase that may help the correct elongation of the peptide intermediate. The present work also revises the structure of heterobactin A that was described by Carrano et al. in 2001. Also, the biochemical characterization of the monooxygenase HMO (encoded by the hmo gene within the gene cluster) established a route for the biosynthesis of the non- proteinogenic amino acid L-hOrn, prior to its incorporation by the NRPS HtbG into the siderophore scaffold. The insights gained from the structural and biochemical characterization of the siderophore heterobactins, together with the genetic and biochemical characterization of the respective biosynthetic gene clusters, allowed us to establish a biosynthetic model for heterobactins assembly. The iron-siderophore binding protein HtbH (encoded by htbH gene within the gene cluster) was also biochemically characterized and was shown to display a novel mix-type catecholate-hydroxamate binding behavior.

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