Characterization of the Aminocoumarin Ligase SimL from the Simocyclinone Pathway and Tandem Incubation with NovM,P,N from the Novobiocin Pathway†

ABSTRACT: Simocyclinone D8 consists of an anguicycline C-glycoside tethered by a tetraene diester linker to an aminocoumarin. Unlike the antibiotics novobiocin, clorobiocin, and coumermycin A1, the phenolic hydroxyl group of the aminocoumarin in simocyclinone is not glycosylated with a decorated noviosyl moiety that is the pharmacophore for targeting bacterial DNA gyrase. We have expressed the Streptomyces antibioticus simocyclinone ligase SimL, purified it from Escherichia coli, and established its ATP-dependent amide bond forming activity with a variety of polyenoic acids including retinoic acid and fumagillin. We have then used the last three enzymes from the novobiocin pathway, NovM, NovP, and NovN, to convert a SimL product to a novel novobiocin analogue, in which the 3-prenyl-4-hydroxybenzoate of novobiocin is replaced with a tetraenoate moiety, to evaluate antibacterial activity. The aminocoumarin antibiotics novobiocin 1, clorobiocin 2, and coumermycin A1 3, produced by various Streptomyces species (Figure 1), contain a bicyclic 3-amino-4,7-dihydroxy-coumarin ring system, which serves as an essential scaffold for targeting them to the bacterial type II topoisomerases DNA gyrase and topoisomerase IV (1-5). Coumermycin A