Three thioesterases are involved in the biosynthesis of phosphinothricin tripeptide in Streptomyces viridochromogenes Tü494.
Phosphinothricin tripeptide (PTT) is a peptide antibiotic produced by Streptomyces viridochromogenes Tü494, and it is synthesized by nonribosomal peptide synthetases. The PTT biosynthetic gene cluster contains three peptide synthetase genes: phsA, phsB, and phsC. Each of these peptide synthetases comprises only one module. In neither PhsB nor PhsC is a typical C-terminal thioesterase domain present. In contrast, a single thioesterase GXSXG motif has been identified in the N terminus of the first peptide synthetase, PhsA. In addition, two external thioesterase genes, theA and theB, are located within the PTT biosynthetic gene cluster. To analyze the thioesterase function as well as the assembly of the peptide synthetases within PTT biosynthesis, several mutants were generated and analyzed. A phsA deletion mutant (MphsA) was complemented with two different phsA constructs that were carrying mutations in the thioesterase motif. In one construct, the thioesterase motif comprising 45 amino acids of phsA were deleted. In the second construct, the conserved serine residue of the GXSXG motif was replaced by an alanine. In both cases, the complementation of MphsA did not restore PTT biosynthesis, revealing that the thioesterase motif in the N terminus of PhsA is required for PTT production. In contrast, TheA and TheB might have editing functions, as an interruption of the theA and theB genes led to reduced PTT production, whereas an overexpression of both genes in the wild type enhanced the PTT yield. The presence of an active single thioesterase motif in the N terminus of PhsA points to a novel mechanism of product release.
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Mikrobiologie/Biotechnologie, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany.
Schwartz
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Wohlleben
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Schinko
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D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D015151:Immunoblotting; D008954:Models, Biological; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism*; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013869:Thiolester Hydrolases / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D009943:Organophosphorus Compounds; C003122:bialaphos; D013869:Thiolester Hydrolases; D010453:Peptide Synthases