The antibiotic compound pyrroindomycin B contains an indole ring chlorinated in the 5 position. The indole ring is probably derived from tryptophan, and thus primers derived from conserved regions of tryptophan halogenases were used to amplify and clone a DNA fragment that was then used to isolate a tryptophan 5-halogenase gene (pyrH) from a cosmid library of the pyrroindomycin producer Streptomyces rugosporus LL-42D005. A gene disruption mutant in the tryptophan 5-halogenase gene no longer produced pyrroindomycin B, but still produced pyrroindomycin A, the nonhalogenated derivative. The halogenase gene could be overexpressed in Pseudomonas fluorescens BL915 DeltaORF1 and was purified to homogeneity by immobilized metal chelate ion affinity chromatography. Chlorinating and brominating activities with tryptophan as a substrate were detected in cell-free extracts and for the purified enzyme.

Salmochelins represent novel carbohydrate containing catecholate siderophores, which are excreted by Salmonella enterica and uropathogenic Escherichia coli strains under low-iron stress. While previous analytical data showed salmochelins to contain 2,3-dihydroxybenzoyl-L-serine and glucose, the molecular structure remained elusive. Structure elucidation with electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry (ESI-FTICR-MS), GC-MS and 2D-NMR now revealed that salmochelins are enterobactin-related compounds, which are beta-C-glucosylated at the 5-position of a 2,3-dihydroxybenzoyl residue. The key compound salmochelin S4 is a twofold beta-C-glucosylated enterobactin analogue. Comparison of partial structures of salmochelin with a C-glycosylated compound previously characterized by another group strongly suggest that salmochelins represent the long sought compounds termed Salmonella resistance factors (SRF) or pacifarins. Transformation of iro-genes into enterobactin-producing E. coli K12 confers the ability to produce salmochelins. A detailed analysis proved iroB to be the sole gene with glycosyltransferase activity necessary for salmochelin production. Salmochelins compared to enterobactin are the better siderophores in the presence of serum albumin. This may indicate for salmochelins a considerably more important role for pathogenic processes in certain Escherichia coli and Salmonella infections than formerly assigned to enterobactin. This conclusion is supported by the location of the iro genes on pathogenicity islands of uropathogenic E. coli strains.

A screening method was established to detect inhibitors of the biosynthetic pathways of aromatic amino acids and para-aminobenzoic acid, the precursor of folic acid, using an agar A successful search for novel antibacterial metabolites has to meet three criteria, first, a specific target which is essential for the metabolism of a bacterium and not yet provided with an known inhibitor. Second, a set of taxonomically characterized and dereplicated microorganisms as producers of secondary metabolites, and last but not least a lucky but experienced hand for strain isolation and cultivation. We have chosen the shikimate pathway as an essential target of bacterial metabolism, with special consideration of the biosynthesis of aromatic amino acids and para-aminobenzoic (pAba) acid derived from the keymetabolite chorismate. Only a few antimetabolites are known as inhibitors of aromatic amino acids, such as L-2,5-dihydrophenylalanine2), an antagonist of phenlyalanine, and glyphosate that inhibits 3-enolpyruvylshikimate-3-phosphate synthase3,4). To our knowledge, no natural product inhibitor of pAba biosynthesis has been described in the literature. This pathway, which is catalyzed by two enzymes, 4-amino-4-deoxychorismic acid (ADC) synthase and ADC lyase, seems to be of considerable interest for the development of novel antibiotics since it is directly linked to folic acid biosynthesis, which is established in plants, fungi, prokaryotes and parasites of the apicomplexa group (Plasmodium, Toxoplasma) but not in vertebrates.As suitable producers of bioactive metabolites we screened within the order Actinomycetales terrestrial and marine members of the families Streptomycetaceae and Micromonosporaceae and rare actinomycete genera. A total of 930 extracts derived from 201 actinomycetes were subjected to the screening. Among them, only AB-18-032, an extract from a marine isolate from a sediment collected from the Sea of Japan, was found to exhibit activity against

Plumbing the depths: Abyssomicin C (structure shown) from ocean floor sediment is a novel antibiotic that inhibits the biosynthesis steps between chorismate and p‐aminobenzoic acid. Its activity may be explained by the irreversible trapping of the targeted enzymes by a Michael addition. Blocking the biosynthesis of p‐aminobenzoic acid may be one approach to developing new antibiotics.

Gene-inactivation studies point to the involvement of OxyB in catalyzing the first oxidative phenol coupling reaction during glycopeptide antibiotic biosynthesis. The oxyB gene has been cloned and sequenced from the vancomycin producer Amycolatopsis orientalis, and the hemoprotein has been produced in Escherichia coli, crystallized, and its structure determined to 1.7-Å resolution. OxyB gave UV-visible spectra characteristic of a P450-like hemoprotein in the low spin ferric state. After reduction to the ferrous state by dithionite or by spinach ferredoxin and ferredoxin reductase, the CO-ligated form gave a 450-nm peak in a UV-difference spectrum. Addition of putative heptapeptide substrates to resting OxyB produced type I changes to the UV spectrum, but no turnover was observed in the presence of ferredoxin and ferredoxin reductase, showing that either the peptides or the reduction system, or both, are insufficient to support a full catalytic cycle. OxyB exhibits the typical P450-fold, with helix L containing the signature sequence FGHGXHXCLG and Cys 347 being the proximal axial thiolate ligand of the heme iron. The structural similarity of OxyB is highest to P450nor, P450terp, CYP119, and P450eryF. In OxyB, the F and G helices are rotated out of the active site compared with P450nor, resulting in a much more open active site, consistent with the larger size of the presumed heptapeptide substrate.

Model mutants. The biosynthesis of glycopeptide antibiotics must be understood before it can be reprogrammed to generate altered antibiotics. Based on a detailed HPLC‐ESI‐MS analysis of linear and cyclic peptide intermediates of balhimycin biosynthesis mutants, a new model for glycopeptide assembly is suggested (see figure). We propose that the three central oxidative cyclizations by P450‐dependant monooxygenases occur during peptide assembly before cleavage from the nonribosomal peptide synthetase complex.

No abstract

In the mutasynthetic approach, the DeltadpgA mutant of the vancomycin-type glycopeptide antibiotic producer Amycolatopsis balhimycina, which is deficient in the synthesis of 3,5-dihydroxyphenylglycine (DPg), was supplemented with synthetic DPg analogues to obtain the corresponding modified glycopeptides. Sterically more demanding 3,5-disubstituted methoxy derivatives as well as monosubstituted DPg analogues were accepted as substrates. These facts indicate that steric and electronic requirements suffice in several cases for the oxidative closure of the AB ring, thus leading to the generation of novel antibiotically active glycopeptide derivatives. The results represent a further step in evaluating the potential of mutasynthesis for peptidic secondary metabolites.