Use of a halogenase of hormaomycin biosynthesis for formation of new clorobiocin analogues with 5-chloropyrrole moieties.

The depsipeptide antibiotic hormaomycin, which is produced by Streptomyces griseoflavus W-384, contains a 5-chloropyrrole moiety. In the producer strain we identified the gene hrmQ that shows sequence similarity to FADH(2)-dependent halogenases. This gene was cloned and heterologously expressed in Streptomyces roseochromogenes var. oscitans DS12.976, which is the producer of the aminocoumarin antibiotic clorobiocin, which contains a 5-methylpyrrole moiety. For the present experiment, we used a mutant of this strain in which the respective pyrrole-5-methyltransferase had been inactivated. Expression of the halogenase hrmQ in this mutant strain led to the formation of two new clorobiocin derivatives that carried a 5-chloropyrrole moiety. These compounds were isolated on a preparative scale, their structures were elucidated by (1)H NMR spectroscopy and mass spectrometry, and their antibacterial activity was determined. The substrate of HrmQ is likely to be a pyrrole-2-carboxyl-S-[acyl carrier protein] thioester. If this assumption is true, this study presents the first experiment in combinatorial biosynthesis that uses a halogenase that acts on an acyl carrier protein-bound substrate.

Biological activities of novel gyrase inhibitors of the aminocoumarin class.

Thirty-one aminocoumarin antibiotics derived from mutasynthesis experiments were investigated for their biological activities. Their inhibitory activities toward Escherichia coli DNA gyrase were determined in two different in vitro assays: an ATPase assay and a DNA supercoiling assay. The assays gave a similar rank order of the activities of the compounds tested, although the absolute 50% inhibitory concentrations (IC(50)s) obtained in each assay were different. To confirm that the compounds also acted as gyrase inhibitors in vivo, reporter gene assays were carried out with E. coli by using gyrA and sulA promoter fusions with the luxCDABE operon. A strong induction of both promoters was observed for those compounds that showed gyrase inhibitory activity in the biochemical assays. Compounds carrying analogs of the prenylated benzoyl moiety (ring A) of clorobiocin that were structurally very different showed high levels of activity both in the biochemical assay and in the reporter gene assay, indicating that the structure of this moiety can be varied considerably without a loss of affinity for bacterial gyrase. The experimentally determined IC(50)s were compared to the binding energies calculated in silico, which indicated that a shift of the pyrrole carboxylic acid moiety from the O-3'' to the O-2'' position of the deoxysugar moiety has a significant impact on the binding mode of the compounds. The aminocoumarin compounds were also investigated for their MICs against different bacterial pathogens. Several compounds showed high levels of activity against staphylococci, including a methicillin-resistant Staphylococcus aureus strain. However, they showed only poor activities against gram-negative strains.

Alternatively activated macrophages regulate extracellular levels of the hormone placental lactogen via receptor-mediated uptake and transcytosis.

Alternatively activated (M2) macrophages regulate immune responses and tissue remodelling. In many tissues including placenta, M2 express stabilin-1, a multidomain protein that exerts a dual role as a scavenger receptor for acetylated low density lipoprotein (acLDL) and SPARC (secreted protein acidic and rich in cysteine) and as an intracellular cargo carrier for SI-CLP. Using yeast two-hybrid screening, we identified the developmental hormone placental lactogen (PL) as a novel ligand of stabilin-1. In Chinese hamster ovary-stabilin-1 cells and M2, FACS and confocal microscopy demonstrated that stabilin-1 mediates internalization and endosomal sorting of PL. In M2 macrophages, PL was partially degraded in lysosomes; part of PL escaped degradation and was delivered to novel PL+ storage vesicles lacking endosomal/lysosomal markers. During formation, PL+ vesicles underwent transient interaction with the trans-Golgi network (TGN). Upon placement of PL-loaded M2 into PL-free medium, PL was secreted into the supernatant. Leupeptin, an inhibitor of lysosomal hydrolases, reduced PL degradation, enhanced sorting of PL into the TGN/storage vesicle pathway and increased PL secretion. Thus, processing of PL in M2 macrophages occurs either by the classical lysosomal pathway or by a novel TGN-associated trans-secretory pathway. Macrophages isolated from human placental villi efficiently endocytosed PL-FITC and transported it to the storage vesicles. Our data show that extracellular PL levels are determined by uptake, degradation, storage, and release in M2. During pregnancy PL concentration reaches 10 microg/ml in maternal circulation and stays below 0.5 microg/ml in fetal circulation. We propose that stabilin-1-positive macrophages determine the difference in PL levels between maternal and fetal circulation.

New aminocoumarin antibiotics derived from 4-hydroxycinnamic acid are formed after heterologous expression of a modified clorobiocin biosynthetic gene cluster.

Three new aminocoumarin antibiotics, termed ferulobiocin, 3-chlorocoumarobiocin and 8'-dechloro-3-chlorocoumarobiocin, were isolated from the culture broth of a Streptomyces coelicolor M512 strain expressing a modified clorobiocin biosynthetic gene cluster. Structural analysis showed that these new aminocoumarins were very similar to clorobiocin, with a substituted 4-hydroxycinnamoyl moieties instead of the prenylated 4-hydroxybenzoyl moiety of clorobiocin. The possible biosynthetic origin of these moieties is discussed.

Improved mutasynthetic approaches for the production of modified aminocoumarin antibiotics.

This study reports improved mutasynthetic approaches for the production of aminocoumarin antibiotics. Previously, the mutasynthetic production of aminocoumarins with differently substituted benzoyl moieties was limited by the substrate specificity of the amide synthetase CloL. We expressed two amide synthetases with different substrate specificity, CouL and SimL, in appropriately engineered producer strains. After feeding of precursor analogs that were not accepted by CloL, but by SimL or CouL, a range of aminocoumarins, unattainable in our previous experiments, was produced and isolated in preparative amounts. Further, we developed a two-stage mutasynthesis procedure for the production of hybrid antibiotics that showed the substitution pattern of novobiocin in the aminocoumarin moiety and that of clorobiocin in the deoxysugar moiety. The substitution pattern of the benzoyl moiety was determined by external addition of an appropriate precursor. Twenty-five aminocoumarin compounds were prepared by these methods, and their structures were elucidated with mass and 1H-NMR spectroscopy.

Biosynthesis of clorobiocin: investigation of the transfer and methylation of the pyrrolyl-2-carboxyl moiety.

Clorobiocin is an aminocoumarin antibiotic containing a 5-methylpyrrolyl-2-carboxyl moiety, attached by an ester bond to a deoxysugar. This pyrrolyl moiety is important for the binding of the antibiotic to its biological target, the B subunit of gyrase. Inactivation experiments had shown that two putative acyl carrier proteins, CloN5 and CloN1, and two putative acyl transferases, CloN2 and CloN7, are involved in the transfer of the pyrrolyl-2-carboxyl moiety to the deoxysugar. In this study, pyrrolyl-2-carboxyl-N-acetylcysteamine thioester was synthesized and fed to cloN1 ( - ), cloN2 ( - ) and cloN7 ( - ) mutants, and secondary metabolite formation was analyzed by HPLC and HPLC-MS. Transfer of the pyrrolyl-2-carboxyl moiety was observed in the cloN1 ( - ) and cloN2 ( - ) mutants, but not in the cloN7 ( - ) mutant, suggesting that CloN7 is responsible for this reaction. The product of this transfer, novclobiocin 109, was not further methylated to the 5-methylpyrrolyl-2-carboxyl compound, i.e. clorobiocin, suggesting that methylation does not take place after the acyl transfer. Additional investigations for the presence of 5-methylpyrrolyl-2-carboxylic acid in the mutants, and inactivation experiments with the methyltransferase gene cloN6, suggested that methylation by CloN6 and acyl transfer by CloN7 take place in a concerted fashion, requiring the presence of both proteins for efficient product formation. A mechanism for the methylation/acyl transfer process in the late steps of clorobiocin biosynthesis, involving CloN1, CloN2, CloN5, CloN6 and CloN7 is suggested.