Study on the biosynthetic mechanism of signalling molecules controlling secondary metabolism in Streptomyces species
Zusammenfassung der Projektergebnisse
y-butyrolactones play an important role in the regulation of antibiotic production and differentiation in Streptomyces. However the biosynthetic pathway for these small molecules has not been determined and in vitro synthesis has riot been reported so far. The function of the AfsA family of proteins, originally proposed to catalyse y-butyrolactone synthesis, has been in debate. To clarify the function of the AfsA family and to understand the synthesis of the y-butyrolactones, we have performed in silico analysis of this protein family. AfsA consist of two divergent domains each of which has similarity to the fatty acid synthesis enzymes FabA and FabZ. Two predicted active sites were mutated in ScbA, the AfsA homologue in the model streptomycete, Streptomyces coelicolor. Theses were the glutamate residue as a single point mutant as well as a double point mutant and the arginine residue. These residues were conserved in all the AfsA family of proteins. The constructed mutants were assessed for the ability to produce y-butyrolactones and none were able to produce the small signalling molecules, strongly suggesting that ScbA has enzymatic activity. The mutation was further introduced to the original position of ScbA in the chromosome. Again the mutants did not produce any y-butyrolactones. However, LW31 possessing two mutated glutamate sites had different antibiotic production profiles in liquid media comparing to the other mutants. These results suggest that ScbA may also posses a regulatory role in antibiotic production. In addition, a new y-butyrolactone detection method, the kanamycin bioassay, utilizing the DNA and the y-butyrolactone binding abilities of the receptor, ScbR was established. This new method was tested for sensitivity compared to the antibiotic bioassay and also used to analyze the ligand specificity determinant. Thirty chemically synthesized racemic autoregulator analogues were tested and ScbR shows the highest affinity toward the natural SCBl. It also shows preference toward a ligand possessing a long C2 side chain (7-10 carbons), a C1'-ß-hydroxyl group and a C6'-methyl group.
Projektbezogene Publikationen (Auswahl)
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ScbA from Streptomyces coelicolor A3(2) has homology to fatty acid synthases and is able to synthesise-butyrolactones. Microbiology (2007) 153:1394-1404
Hsaio, N.H., Soeding J, Linke D, Lange C, Hertweck C, Wohlleben W, Takano E
