title: Biophysical investigation of the mode of inhibition of tetramic acids, the allosteric inhibitors of undecaprenyl pyrophosphate synthase.
abstract: Undecaprenyl pyrophosphate synthase (UPPS) catalyzes the consecutive condensation of eight molecules of isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate the C(55) undecaprenyl pyrophosphate (UPP). It has been demonstrated that tetramic acids (TAs) are selective and potent inhibitors of UPPS, but the mode of inhibition was unclear. In this work, we used a fluorescent FPP probe to study possible TA binding at the FPP binding site. A photosensitive TA analogue was designed and synthesized for the study of the site of interaction of TA with UPPS using photo-cross-linking and mass spectrometry. The interaction of substrates with UPPS and with the UPPS.TA complex was investigated by protein fluorescence spectroscopy. Our results suggested that tetramic acid binds to UPPS at an allosteric site adjacent to the FPP binding site. TA binds to free UPPS enzyme but not to substrate-bound UPPS. Unlike Escherichia coli UPPS which follows an ordered substrate binding mechanism, Streptococcus pneumoniae UPPS appears to follow a random-sequential substrate binding mechanism. Only one substrate, FPP or IPP, is able to bind to the UPPS.TA complex, but the quaternary complex, UPPS.TA.FPP.IPP, cannot be formed. We propose that binding of TA to UPPS significantly alters the conformation of UPPS needed for proper substrate binding. As the result, substrate turnover is prevented, leading to the inhibition of UPPS catalytic activity. These probe compounds and biophysical assays also allowed us to quickly study the mode of inhibition of other UPPS inhibitors identified from a high-throughput screening and inhibitors produced from a medicinal chemistry program.
authors.affiliation: Infectious Diseases, Novartis Institutes for BioMedical Research, Inc., Cambridge, Massachusetts 02139, USA.
authors.forename: Lac V
authors.initials: LV
authors.lastname: Lee
authors.forename: Brian
authors.initials: B
authors.lastname: Granda
authors.forename: Karl
authors.initials: K
authors.lastname: Dean
authors.forename: Jianshi
authors.initials: J
authors.lastname: Tao
authors.forename: Eugene
authors.initials: E
authors.lastname: Liu
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.forename: Stefan
authors.initials: S
authors.lastname: Peukert
authors.forename: Sompong
authors.initials: S
authors.lastname: Wattanasin
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Xie
authors.forename: Neil S
authors.initials: NS
authors.lastname: Ryder
authors.forename: Ruben
authors.initials: R
authors.lastname: Tommasi
authors.forename: Gejing
authors.initials: G
authors.lastname: Deng
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001703:Biophysics; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology; D005456:Fluorescent Dyes; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011760:Pyrrolidinones / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D021241:Spectrometry, Mass, Electrospray Ionization; D013296:Streptococcus pneumoniae / Q000201:enzymology
chemical_list: D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D011760:Pyrrolidinones; C009435:tetramic acid; D019883:Alkyl and Aryl Transferases; C023266:undecaprenyl pyrophosphate synthetase