Biochemistry and Cell Biology Department, Stony Brook Universi...

Viridicatumtoxins are a rare class of tetracycline-like antibiotics that strongly inhibit drug-resistant Gram-positive bacteria Although reported to exhibit in vitro inhibition activity to undecaprenyl pyrophosphate synthase UPPS an essential enzyme in bacterial cell wall synthesis the biological targets and mechanism of action of viridicatumtoxins especially the drug-target interactions remain largely unknown In this study the structure of Enterococcus faecalis UPPS EfaUPPS was first determined uncovering that EfaUPPS can form not only a typical functional dimer but also an unexpected atypical dimer We then observed that viridicatumtoxins A VirA and B VirB are able to bind to UPPSs of E faecalis S aureus and E coli in a direct and high-affinity manner as evidenced by in vitro enzyme inhibition assay surface plasmon resonance SPR binding analysis and in vivo growth inhibition assay demonstrating that viridicatumtoxins exert antibacterial effects through UPPS binding The key amino acid residues involved in the interactions with VirA and VirB in EfaUPPS binding pocket were revealed by molecular docking studies and further validated by site-directed mutagenesis A single mutation of EfaUPPS at D29A N31A and R42A can obviously increase their affinities to VirA while a single mutation at W228A conferred significant resistance to VirA Moreover translation inhibition assay showed that VirA and VirB can weakly inhibit E coli 70S ribosome The weak inhibition of ribosome was proposed to be attributed to steric hindrance between viridicatumtoxin ring F and 70S ribosome helix 34 by molecular docking study Our structural biochemical and computational investigations on the interactions of viridicatumtoxins with UPPS and 70S ribosome not only disclosed the potential biological targets of viridicatumtoxins but also provided a theoretical basis for structural optimization to make new viridicatumtoxin derivatives with improved antimicrobial activities

Biochemistry and Cell Biology Department, Stony Brook University, Stony Brook, New York 11794, United States.

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Investigations into the Antibacterial Mechanism of Action of Viridicatumtoxins.