An unusual amide synthetase (CouL) from the coumermycin A1 biosynthetic gene cluster from Streptomyces rishiriensis DSM 40489.
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.
Schmutz
Elisabeth
E
Eberhard-Karls-Universität Tübingen, Pharmazeutische Biologie, Tübingen, Germany Institut für Pflanzenbiochemie, Halle (Saale), Germany.
Steffensky
Marion
M
Schmidt
Jürgen
J
Porzel
Andrea
A
Li
Shu-Ming
SM
Heide
Lutz
L
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
D049933:Aminocoumarins; D003374:Coumarins; D009097:Multienzyme Complexes; D009675:Novobiocin; C006260:clorobiocin; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; C004628:coumermycin