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

Abstract

Simocyclinone D 8 consists of an anguicycline C-glycoside tethered by a tetraene diester linker to an aminocoumarin. Unlike the antibiotics novobiocin, clorobiocin, and coumermycin A 1 , 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 A 1 3, produced by various Streptomyces species (Figure 1), contain a bicyclic 3-amino-4,7-dihydroxycoumarin 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 1 is a pseudosymmetric dimer, containing elements of novobiocin (the 8-methylaminocoumarin) and clorobiocin (the 3′-O-methylpyrrolyl acyl group). Cocrystals of the N-terminal 24 kDa subfragment of the DNA gyrase B subunit (GyrB) 1 with 1 and 2 (1, 2) reveal that this family of antibiotics uses the aminocoumarin as a planar scaffold to present the decorated 4′-O-methyl-3′-O-acyl noviosyl moiety as the pharmacophore for inhibiting ATP hydrolysis in GyrB. These decorations of the L-deoxy sugar noviose include 4′-Omethylation and 3′-O-acylation with either a carbamoyl or methylpyrrolyl group. This 3′-O-acyl substituent is in intimate contact with GyrB active site residues and/or bound water molecules.Recently, another antibiotic containing the conserved aminocoumarin moiety, simocyclinone D 4 from Streptomyces antibioticus, has been discovered (6, 7). Simocyclinone D is a structurally unique antibiotic containing an aromatic anguicycline polyketide nucleus, the deoxy sugar D-olivose, and either the 8-chloro or 8-desmethyl version of the 3-amino-4,7-dihydroxycoumarin (8). The anguicycline Cglycoside and the aminocoumarin elements are linked by a tetraene dicarboxylic acid moiety. Simocyclinone possesses antimicrobial activity against Gram-positive bacteria, as well as exhibiting cytostatic effects against human tumor cell lines (6). However, its lack of a decorated noviosyl moiety at the 7-hydroxy position of the aminocoumarin scaffold suggests a molecular mechanism of action different than that of 1 and 2 given the X-ray information on GyrB complexes noted above for 1 and 2 (1, 2). Indeed, recent studies reveal that simocyclinone D8 is a potent inhibitor of gyrase, albeit through a novel mode of action by preventing the initial binding of gyrase to DNA (Anthony Maxwell, personal communicatio...

The evolution of gene collectives: How natural selection drives chemical innovation

“…In all likelihood, the linkage of these three subclusters within a single genome triggered the ''invention'' of the hybrid small molecule simocyclinone. An important avenue of inquiry for simocyclinone (as for glycosyltransferases from aminoglycoside gene clusters) implicates the enzymes that link the three chemical groups together (83,84). Because these conjugating enzymes are necessary for the functioning of a new ''supercluster,'' but would not have been required for the original gene clusters, their evolutionary origin is unclear.…”

Biochemical and Genetic Insights into Asukamycin Biosynthesis

“…AsuD3, an acyl-CoA ligase, is proposed to act in the C 5 N ring formation, since disruption of the homologous moeA4 in Streptomyces ghanaensis led to accumulation of a moenomycin A analog lacking the C 5 N moiety (14). AsuD1 is closely related to a group of amide synthases, including SimL for simocyclinone biosynthesis (43,44). All of the asuD1, D2, and D3 mutants failed to produce asukamycin but accumulated a new set of metabolites, A1a-A5a, which carry a free carboxyl group in place of the amide-linked C 5 N moiety of A1-A5, respectively (supplemental Fig.…”

Adenylate‐Forming Enzymes of Rubradirin Biosynthesis: RubC1 Is a Bifunctional Enzyme with Aminocoumarin Acyl Ligase and Tyrosine‐Activating Domains

“…In this study, we have shown that Orf4 is an active aminocoumarin acyl ligase with biochemical properties similar to those of SimL of simocyclinone biosynthesis. [23,31] However, RubF6, which shares 88 % identity with Orf4 on the amino acid level, was inactive. Site-directed mutagenesis of the active site allowed us to convert RubF6 into an active aminocoumarin acyl ligase.…”