TAILIEUCHUNG - Báo cáo khoa học: An unusual amide synthetase (CouL) from the coumermycin A1 biosynthetic gene cluster from Streptomyces rishiriensis DSM 40489

The aminocoumarin antibiotic coumermycin A1produced byStreptomyces rishiriensisDSM40489 contains two amide bonds. The biosynthetic gene cluster ofcoumermycin con-tains a putative amide synthetase gene, couL, encoding a protein of529 amino acids. CouL was overexpressed as hexahistidine fusion protein inEscherichia coliand purified by metal affinity chromatography, resulting in a nearly homogenous protein. | Eur. J. Biochem. 270 4413-4419 2003 FEBS 2003 doi An unusual amide synthetase CouL from the coumermycin A1 biosynthetic gene cluster from Streptomyces rishiriensis DSM 40489 Elisabeth Schmutz1 Marion Steffensky1 Jurgen Schmidt2 Andrea Porzel2 Shu-Ming Li1 and Lutz Heide1 1 Eberhard-Karls-Universitat Tubingen Pharmazeutische Biologie Tubingen Germany 2Institutfur Pflanzenbiochemie Halle Saale Germany The aminocoumarin antibiotic coumermycin A1 produced by Streptomyces rishiriensis DSM 40489 contains two amide bonds. The biosynthetic gene cluster of roumermycin contains a putative amide synthetase gene couL encoding a protein of 529 mténo ccids. CouL was vvereppressed as hexahistidine fusion protein in Escherichia coli and purified by metal affinity chromatography resulting in a nearly homogenous protein. CouU catalysed the formation of both amide bonds of a1unsem ycm A1 . between the central 3-methylpyrrole-2 4-dicarboxylic acid and two aminocoumarin moieties. Gel exclusion chromatography showed that the enzyme is active as a monomer. The activity was strictly dependent on the presence of ATP and Mil2 or Mg2 . The apparent Km values were determined as 26 M for the 3-mrthylpyeeolr-2 4-dicarboxylic acid and 44 M for the aminocoumarin moiety respectively. Several analogues ofthe pyrrole dicarboxylic acid were accepted as substrates. In contrast pyridine carboxylic acids were not accepted. 3-Dimethylallyl-4-hydroxybenzoic acid the acyl component in novobiocin biosynthesis was well accepted despite its structural difference from the genuine acyl substrate of Couu. Keywords amide synthetase aminocoumarin antibiotic biosynthesis um . Coumrpmycin A1 like novobiocin and clorobiocin belongs to the aminocoumarin antibiotics and is produced by Streptomyces rishiriensis DSM 40482. These antibiotics are potent inhibitors of bacterial DNA gyrase. Their inseraciion with the ATP-binding site in the B subunit of the en yme has been investigated by

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