Overexpression, purification and characterization of SimL, an amide synthetase involved in simocyclinone biosynthesis.
Simocyclinone D8 is a potent inhibitor of bacterial gyrase, produced by Streptomyces antibioticus Tu 6040. It contains an aminocoumarin moiety, similar to that of novobiocin, which is linked by an amide bond to a structurally complex acyl moiety, consisting of an aromatic angucycline polyketide nucleus, the deoxysugar olivose and a tetraene dicarboxylic acid. We have now investigated the enzyme SimL, responsible for the formation of the amide bond of simocyclinone. The gene was cloned, expressed in S. lividans T7, and the protein was purified to near homogeneity, and characterized. The 60 kDa protein catalyzed both the ATP-dependent activation of the acyl component as well as its transfer to the amino group of the aminocoumarin ring, with no requirement for a 4'-phosphopantetheinyl cofactor. Besides its natural substrate, simocyclinone C4, SimL also accepted a range of cinnamic and benzoic acid derivatives and several other, structurally very diverse acids. These findings make SimL a possible tool for the creation of new aminocoumarin antibiotics.
Luft
Thomas
T
Pharmazeutische Biologie, Pharmazeutisches Institut, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, 72076, Tübingen, Germany.
Li
Shu-Ming
SM
Scheible
Holger
H
Kammerer
Bernd
B
Heide
Lutz
L
D000255:Adenosine Triphosphate / Q000378:metabolism; D019732:Amide Synthases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003067:Coenzymes / Q000494:pharmacology; D003374:Coumarins / Q000378:metabolism; D006027:Glycosides / Q000096:biosynthesis; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013303:Streptomyces antibioticus / Q000201:enzymology* / Q000235:genetics; D048170:Streptomyces lividans / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D003067:Coenzymes; D003374:Coumarins; D006027:Glycosides; D011994:Recombinant Proteins; C416814:simocyclinone D8; D000255:Adenosine Triphosphate; D019732:Amide Synthases