clorobiocin and 2-pyrrolecarboxylic-acid

Clorobiocin is an aminocoumarin antibiotic containing a pyrrole-2-carboxyl moiety, attached through an ester bond to a deoxysugar. The pyrrole moiety is important for the binding of the antibiotic to its biological target, gyrase. The complete biosynthetic gene cluster for clorobiocin has been cloned and sequenced from the natural producer, Streptomyces roseochromogenes DS 12.976. In this study, the genes cloN1 and cloN7 were deleted separately from a cosmid containing the complete clorobiocin cluster. The modified cosmids were introduced into the genome of the heterologous host Streptomyces coelicolor M512 by using the integration functions of the PhiC31 phage. While a heterologous producer strain harbouring the intact clorobiocin biosynthetic gene cluster accumulated clorobiocin, the cloN1- and cloN7-defective integration mutants accumulated a clorobiocin derivative that lacked the pyrrole-2-carboxyl moiety, while also producing free pyrrole-2-carboxylic acid. The structures of these metabolites were confirmed by NMR and MS analysis. These results showed that CloN1 and CloN7, together with the previously investigated CloN2, are involved in the transfer of the pyrrole-2-carboxyl moiety to the deoxysugar of clorobiocin. A possible mechanism for the role of these three proteins in the acyl-transfer process is suggested.

Topics: Acylation; Carbohydrates; Cosmids; Multienzyme Complexes; Novobiocin; Proline; Streptomyces

The aminocoumarin antibiotic clorobiocin contains a 5-methylpyrrole-2-carboxylic acid unit. This pyrrole unit is derived from L-proline, and it would be expected that its 5-methyl group should be introduced by a methylation reaction. However, sequence analysis of the clorobiocin biosynthetic gene cluster did not reveal a gene with sequence similarity to the SAM-dependent methyltransferases that could be assigned to this reaction. This study, however, has provided evidence that the gene cloN6 is involved in this methylation reaction. Its gene product CloN6 shares conserved sequence motifs with the recently identified radical SAM protein superfamily, and it has been suggested that members of this family can catalyse methylcobalamin-dependent methylation reactions. cloN6 was inactivated in the clorobiocin producer Streptomyces roseochromogenes var. oscitans DS 12.976 by use of the PCR-targeting method. The cloN6(-) mutants accumulated, instead of clorobiocin, a derivative lacking the 5"'-methyl group of the pyrrole moiety (termed novclobiocin 109). A structural isomer carrying the pyrrole-2-carboxyl moiety at 2"-OH rather than at the 3"-OH of the deoxysugar (novclobiocin 110), and a derivative completely lacking the pyrrole unit (novclobiocin 104) were also identified. The structures of the metabolites were confirmed by NMR and MS analysis. Antibacterial activity tests against Bacillus subtilis showed that novclobiocin 109 and novclobiocin 110 have antibacterial activities about eight times less than that of clorobiocin, whereas novclobiocin 104 showed no activity under the test conditions.

Topics: Anti-Bacterial Agents; Bacillus subtilis; Binding Sites; Chromatography, Liquid; Cloning, Molecular; Genes, Bacterial; Mass Spectrometry; Mutation; Novobiocin; Proline; Protein Methyltransferases; Streptomyces

The aminocoumarin antibiotic clorobiocin contains a 5-methylpyrrole-2-carboxylic acid unit, attached via an ester bond to the 3-OH group of the deoxysugar moiety. To investigate candidate genes responsible for the formation of this ester bond, a gene inactivation experiment was carried out in the clorobiocin producer Streptomyces roseochromogenes var. oscitans DS 12.976. An in-frame deletion was created in the coding sequence of the gene cloN2. The production of secondary metabolites in the wild-type and in the cloN2 mutant was analysed. The wild-type showed clorobiocin as the main product, whereas the cloN2 mutant accumulated a new aminocoumarin derivative, novclobiocin 104, lacking the pyrrole moiety at the 3-OH of the deoxysugar. In addition, free pyrrole-2-carboxylic acid accumulated in the culture extract of the cloN2 mutant. The structures of the metabolites were confirmed by NMR and LC-MS analysis. Clorobiocin production was successfully restored in the cloN2 mutant by introducing a replicative plasmid containing the cloN2 sequence. These results prove an involvement of cloN2 in the formation of the ester bond between the pyrrole moiety and the deoxysugar in clorobiocin biosynthesis. Furthermore, they indicate that the C-methylation at position 5 of the pyrrole moiety occurs after the attachment of pyrrole-2-carboxylic acid unit to the deoxysugar moiety.

Topics: Acyltransferases; Anti-Bacterial Agents; Binding Sites; Chromatography, Liquid; Gene Deletion; Genes, Bacterial; Genetic Complementation Test; Magnetic Resonance Spectroscopy; Mass Spectrometry; Models, Chemical; Molecular Structure; Mutation; Novobiocin; Proline; Streptomyces