Abstract
The aminocoumarin antibiotic coumermycin A1 produced by Streptomyces rishiriensis DSM 40489 contains two amide bonds. The biosynthetic gene cluster of coumermycin contains a putative amide synthetase gene, couL, encoding a protein of 529 amino acids. CouL was overexpressed as hexahistidine fusion protein in Escherichia coli and purified by metal affinity chromatography, resulting in a nearly homogenous protein. CouL catalysed the formation of both amide bonds of coumermycin A1, i.e. 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 Mn2+ or Mg2+. The apparent Km values were determined as 26 micro m for the 3-methylpyrrole-2,4-dicarboxylic acid and 44 micro m for the aminocoumarin moiety, respectively. Several analogues of the 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 CouL.
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