ABSTRACT
Nonribosomal peptides contain a wide range of unusual non-proteinogenic amino acid residues. As a result, these complex natural products are amongst the most structurally diverse secondary metabolites in nature, and possess a broad spectrum of biological activities. beta-Hydroxylation of amino acid precursors or peptidyl residues and their subsequent processing by downstream tailoring enzymes are some of the most common themes in the biosynthetic diversification of these therapeutically important peptides. Identification and characterization of the biosynthetic intermediates and enzymes involved in these processes are thus pivotal in understanding nonribosomal peptide assembly and modification. To this end, the putative asparaginyl oxygenase- and 3-hydroxyasparaginyl phosphotransferase-encoding genes hasP and asnO were separately deleted from the calcium-dependent antibiotic (CDA) biosynthetic gene cluster of Streptomyces coelicolor. Whilst the parent strains produce a number of 3-hydroxyasparagine- and 3-phosphohydroxyasparagine-containing CDAs, the DeltahasP mutants produce exclusively non-phosphorylated CDAs. On the other hand, DeltaasnO mutants produce several new Asn-containing CDAs not present in the wild-type, which retain calcium-dependent antimicrobial activity. This confirms that AsnO and HasP are required for the beta-hydroxylation and phosphorylation of the Asn residue within CDA.
Subject(s)
Asparagine/metabolism , Oxygenases/metabolism , Peptide Biosynthesis, Nucleic Acid-Independent , Peptides/metabolism , Phosphotransferases/metabolism , Streptomyces coelicolor/metabolism , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Antibiosis , Bacillus/drug effects , Gene Deletion , Mass Spectrometry , Microbial Sensitivity Tests , Micrococcus luteus/drug effects , Molecular Structure , Oxygenases/genetics , Peptides/chemistry , Phosphotransferases/genetics , Streptomyces coelicolor/geneticsABSTRACT
The calcium-dependent antibiotic (CDA), from Streptomyces coelicolor, is an acidic lipopeptide comprising an N-terminal 2,3-epoxyhexanoyl fatty acid side chain and several nonproteinogenic amino acid residues. S. coelicolor grown on solid media was shown to produce several previously uncharacterized peptides with C-terminal Z-dehydrotryptophan residues. The CDA biosynthetic gene cluster contains open reading frames encoding nonribosomal peptide synthetases, fatty acid synthases, and enzymes involved in precursor supply and tailoring of the nascent peptide. On the basis of protein sequence similarity and chemical reasoning, the biosynthesis of CDA is rationalized. Deletion of SCO3229 (hmaS), a putative 4-hydroxymandelic acid synthase-encoding gene, abolishes CDA production. The exogenous supply of 4-hydroxymandelate, 4-hydroxyphenylglyoxylate, or 4-hydroxyphenylglycine re-establishes CDA production by the DeltahmaS mutant. Feeding analogs of these precursors to the mutant resulted in the directed biosynthesis of novel lipopeptides with modified arylglycine residues.
