title: An unusual amide synthetase (CouL) from the coumermycin A1 biosynthetic gene cluster from Streptomyces rishiriensis DSM 40489.
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.
authors: Eberhard-Karls-Universität Tübingen, Pharmazeutische Biologie, Tübingen, Germany Institut für Pflanzenbiochemie, Halle (Saale), Germany.
authors: Elisabeth
authors: E
authors: Schmutz
authors: Marion
authors: M
authors: Steffensky
authors: Jürgen
authors: J
authors: Schmidt
authors: Andrea
authors: A
authors: Porzel
authors: Shu-Ming
authors: SM
authors: Li
authors: Lutz
authors: L
authors: Heide
mesh_terms: D000595:Amino Acid Sequence; D049933:Aminocoumarins; D003001:Cloning, Molecular; D003374:Coumarins / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009675:Novobiocin / Q000031:analogs & derivatives* / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D049933:Aminocoumarins; D003374:Coumarins; D009097:Multienzyme Complexes; D009675:Novobiocin; C006260:clorobiocin; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; C004628:coumermycin