Bifunctional Nitrone-Conjugated Secondary Metabolite Targeting the Ribosome.
Emilianne M Limbrick, Michael Graf, Dagmara K Derewacz, Fabian Nguyen, Jeffrey M Spraggins, Maximiliane Wieland, Audrey E Ynigez-Gutierrez, Benjamin J Reisman, Boris Zinshteyn, Kathryn M McCulloch, T M Iverson, Rachel Green, Daniel N Wilson, Brian O Bachmann
Author Information
Emilianne M Limbrick: Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37235, United States.
Michael Graf: Institute of Biochemistry and Molecular Biology, University of Hamburg, Hamburg 20146, Germany.
Dagmara K Derewacz: Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37235, United States.
Fabian Nguyen: Department of Biochemistry, University of Munich, 81377 Munich, Germany.
Jeffrey M Spraggins: Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37235, United States. ORCID
Maximiliane Wieland: Institute of Biochemistry and Molecular Biology, University of Hamburg, Hamburg 20146, Germany.
Audrey E Ynigez-Gutierrez: Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37235, United States.
Benjamin J Reisman: Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37235, United States.
Boris Zinshteyn: Department of Molecular Biology and Genetics, Johns Hopkins University. Baltimore, Maryland 21205, United States.
Kathryn M McCulloch: Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, United States. ORCID
T M Iverson: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37205, United States.
Rachel Green: Department of Molecular Biology and Genetics, Johns Hopkins University. Baltimore, Maryland 21205, United States.
Daniel N Wilson: Institute of Biochemistry and Molecular Biology, University of Hamburg, Hamburg 20146, Germany. ORCID
Brian O Bachmann: Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37235, United States. ORCID
Many microorganisms possess the capacity for producing multiple antibiotic secondary metabolites. In a few notable cases, combinations of secondary metabolites produced by the same organism are used in important combination therapies for treatment of drug-resistant bacterial infections. However, examples of conjoined roles of bioactive metabolites produced by the same organism remain uncommon. During our genetic functional analysis of oxidase-encoding genes in the everninomicin producer var. , we discovered previously uncharacterized antibiotics everninomicin N and O, comprised of an everninomicin fragment conjugated to the macrolide rosamicin via a rare nitrone moiety. These metabolites were determined to be hydrolysis products of everninomicin P, a nitrone-linked conjugate likely the result of nonenzymatic condensation of the rosamicin aldehyde and the octasaccharide everninomicin F, possessing a hydroxylamino sugar moiety. Rosamicin binds the erythromycin macrolide binding site approximately 60 �� from the orthosomycin binding site of everninomicins. However, while individual ribosomal binding sites for each functional half of everninomicin P are too distant for bidentate binding, ligand displacement studies demonstrated that everninomicin P competes with rosamicin for ribosomal binding. Chemical protection studies and structural analysis of everninomicin P revealed that everninomicin P occupies both the macrolide- and orthosomycin-binding sites on the 70S ribosome. Moreover, resistance mutations within each binding site were overcome by the inhibition of the opposite functional antibiotic moiety binding site. These data together demonstrate a strategy for coupling orthogonal antibiotic pharmacophores, a surprising tolerance for substantial covalent modification of each antibiotic, and a potential beneficial strategy to combat antibiotic resistance.
