Antibacterial Mechanism Of Viridicatumtoxin Compounds Targeting UPP Synthase

Viridicatumtoxins are a rare class of tetracycline-like antibiotics that strongly inhibit drugresistant Gram-positive bacteria.Despite reported in vitro inhibitory activity against undecylenyl pyrophosphate synthase(UPPS),an essential enzyme in bacterial cell wall synthesis,the biological targets and mechanisms of action of Viridicatumtoxin,especially drug-target interactions The effect remains largely unknown.In this study,we constructed the recombinant plasmid EfaUPPS-pET28a and transformed into E.coli strain BL21(DE3)to express and purify the high-purity EfaUPPS protein.and the structure of Enterococcus faecalis UPPS(EfaUPPS)was first determined.Then,we observed that Viridicatumtoxin A(VirA)and B(VirB)could bind to UPPS of Enterococcus faecalis in a direct and high-affinity manner with IC50 values of in vitro enzyme inhibition assays,respectively 1.6±0.04 μM(VirA)and 58.1±0.01 nM(VirB),the equilibrium ionization constants(KD)of VirA and VirB combined with EfaUPPS obtained by surface plasmon resonance(SPR)were 123μM and 11.5 μM,respectively.It is notable that the changes in IC50-MT/IC50-WT of EfaUPPS mutants may be attributed to the changes of binding affinity to FPP.Considering this possibility,we tried to acquire Michaelis-Menten parameters,and the results were basically consistent with the previous experimental conclusions.Combined with in vivo growth inhibition experiments,it is demonstrated that Viridicatumtoxin 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.In summary,our structural,biochemical,and computational studies of the interaction of Viridicatumtoxin with UPPS not only reveal potential biological targets of Viridicatumtoxin,but also provide structural optimization for the preparation of new Viridicatumtoxin derivatives with higher antibacterial activity.provides a theoretical basis.