Biophysical Investigation of the Mode of Inhibition of Tetramic Acids, the Allosteric Inhibitors of Undecaprenyl Pyrophosphate Synthase

摘要

Undecaprenyl pyrophosphate synthase (UPPS) catalyzes the consecutive condensation of eight mole-ncules of isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate the C55 undecaprenylnpyrophosphate (UPP). It has been demonstrated that tetramic acids (TAs) are selective and potent inhibitors ofnUPPS, but the mode of inhibition was unclear. In this work, we used a fluorescent FPP probe to study possible TAnbinding at the FPP binding site.Aphotosensitive TAanaloguewas designed and synthesized for the study of the sitenof interaction of TA with UPPS using photo-cross-linking and mass spectrometry. The interaction of substratesnwith UPPS and with the UPPS 3nTA complex was investigated by protein fluorescence spectroscopy. Our resultsnsuggested that tetramic acid binds to UPPS at an allosteric site adjacent to the FPP binding site. TA binds to freenUPPS enzyme but not to substrate-boundUPPS.Unlike Escherichia coliUPPS which follows an ordered substratenbinding mechanism, Streptococcus pneumoniae UPPS appears to follow a random-sequential substrate bindingnmechanism.Only one substrate, FPP or IPP, is able to bind to theUPPS 3nTAcomplex, but the quaternary complex,nUPPS 3nTA3nFPP3nIPP, cannot be formed. We propose that binding of TA to UPPS significantly alters thenconformation of UPPS needed for proper substrate binding. As the result, substrate turnover is prevented, leadingnto the inhibition of UPPS catalytic activity. These probe compounds and biophysical assays also allowed us tonquickly study the mode of inhibition of other UPPS inhibitors identified from a high-throughput screening andninhibitors produced from a medicinal chemistry program.