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Structure of undecaprenyl pyrophosphate synthase from Acinetobacter baumannii.

ABSTRACT: Undecaprenyl pyrophosphate (UPP) is an important carrier of the oligosaccharide component in peptidoglycan synthesis. Inhibition of UPP synthase (UPPS) may be an effective strategy in combating the pathogen Acinetobacter baumannii, which has evolved to be multidrug-resistant. Here, A. baumannii UPPS (AbUPPS) was cloned, expressed, purified and crystallized, and its structure was determined by X-ray diffraction. Each chain of the dimeric protein folds into a central ?-sheet with several surrounding ?-helices, including one at the C-terminus. In the active site, two molecules of citrate interact with the side chains of the catalytic aspartate and serine. These observations may provide a structural basis for inhibitor design against AbUPPS.

SUBMITTER: Ko TP

PROVIDER: S-EPMC6277960 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Structure of undecaprenyl pyrophosphate synthase from Acinetobacter baumannii.

Ko Tzu Ping TP Huang Chi Hung CH Lai Shu Jung SJ Chen Yeh Y

Acta crystallographica. Section F, Structural biology communications 20181116 Pt 12

Undecaprenyl pyrophosphate (UPP) is an important carrier of the oligosaccharide component in peptidoglycan synthesis. Inhibition of UPP synthase (UPPS) may be an effective strategy in combating the pathogen Acinetobacter baumannii, which has evolved to be multidrug-resistant. Here, A. baumannii UPPS (AbUPPS) was cloned, expressed, purified and crystallized, and its structure was determined by X-ray diffraction. Each chain of the dimeric protein folds into a central β-sheet with several surroundi ...[more]

PMID: 30511669

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Cocktailed fragment screening by X-ray crystallography of the antibacterial target undecaprenyl pyrophosphate synthase from Acinetobacter baumannii.

Project description:Direct soaking of protein crystals with small-molecule fragments grouped into complementary clusters is a useful technique when assessing the potential of a new crystal system to support structure-guided drug discovery. It provides a robustness check prior to any extensive crystal screening, a double check for assay binding cutoffs and structural data for binding pockets that may or may not be picked out in assay measurements. The structural output from this technique for three novel fragment molecules identified to bind to the antibacterial target Acinetobacter baumannii undecaprenyl pyrophosphate synthase are reported, and the different physicochemical requirements of a successful antibiotic are compared with traditional medicines.

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Substrate and product specificities of cis-type undecaprenyl pyrophosphate synthase.

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Species differences in alternative substrate utilization by the antibacterial target undecaprenyl pyrophosphate synthase.

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Structure of ATP synthase from ESKAPE pathogen <i>Acinetobacter baumannii</i>.

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Crystal structure of undecaprenyl-pyrophosphate phosphatase and its role in peptidoglycan biosynthesis.

Project description:As a protective envelope surrounding the bacterial cell, the peptidoglycan sacculus is a site of vulnerability and an antibiotic target. Peptidoglycan components, assembled in the cytoplasm, are shuttled across the membrane in a cycle that uses undecaprenyl-phosphate. A product of peptidoglycan synthesis, undecaprenyl-pyrophosphate, is converted to undecaprenyl-phosphate for reuse in the cycle by the membrane integral pyrophosphatase, BacA. To understand how BacA functions, we determine its crystal structure at 2.6 Å resolution. The enzyme is open to the periplasm and to the periplasmic leaflet via a pocket that extends into the membrane. Conserved residues map to the pocket where pyrophosphorolysis occurs. BacA incorporates an interdigitated inverted topology repeat, a topology type thus far only reported in transporters and channels. This unique topology raises issues regarding the ancestry of BacA, the possibility that BacA has alternate active sites on either side of the membrane and its possible function as a flippase.

| S-EPMC5852022 | biostudies-literature

Reducing the Level of Undecaprenyl Pyrophosphate Synthase Has Complex Effects on Susceptibility to Cell Wall Antibiotics.

Project description:Undecaprenyl pyrophosphate synthase (UppS) catalyzes the formation of the C55 lipid carrier (UPP) that is essential for bacterial peptidoglycan biosynthesis. We selected here a vancomycin (VAN)-resistant derivative of Bacillus subtilis W168 that contains a single-point mutation in the ribosome-binding site of the uppS gene designated uppS1 Genetic reconstruction experiments demonstrate that the uppS1 allele is sufficient to confer low-level VAN resistance and causes reduced UppS translation. The decreased level of UppS renders B. subtilis slightly more susceptible to many late-acting cell wall antibiotics, including ?-lactams, but significantly more resistant to fosfomycin and d-cycloserine, antibiotics that interfere with the very early steps of cell wall synthesis. We further show that the uppS1 allele leads to slightly elevated expression of the ?M regulon, possibly helping to compensate for the stress caused by a decrease in UPP levels. Notably, the uppS1 mutation increases resistance to VAN, fosfomycin, and d-cycloserine in wild-type cells, but this effect is greatly reduced or eliminated in a sigM mutant background. Our findings suggest that, although UppS is an attractive antibacterial target, incomplete inhibition of UppS function may lead to increased resistance to some cell wall-active antibiotics.

| S-EPMC3754353 | biostudies-literature

Anthranilic Acid Inhibitors of Undecaprenyl Pyrophosphate Synthase (UppS), an Essential Enzyme for Bacterial Cell Wall Biosynthesis.

Project description:We report the successful implementation of virtual screening in the discovery of new inhibitors of undecaprenyl pyrophosphate synthase (UppS) from Escherichia coli. UppS is an essential enzyme in the biosynthesis of bacterial cell wall. It catalyzes the condensation of farnesyl pyrophosphate (FPP) with eight consecutive isopentenyl pyrophosphate units (IPP), in which new cis-double bonds are formed, to generate undecaprenyl pyrophosphate. The latter serves as a lipid carrier for peptidoglycan synthesis, thus representing an important target in the antibacterial drug design. A pharmacophore model was designed on a known bisphosphonate BPH-629 and used to prepare an enriched compound library that was further docked into UppS conformational ensemble generated by molecular dynamics experiment. The docking resulted in three anthranilic acid derivatives with promising inhibitory activity against UppS. Compound 2 displayed high inhibitory potency (IC50 = 25 μM) and good antibacterial activity against E. coli BW25113 ΔtolC strain (MIC = 0.5 μg/mL).

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Biophysical investigation of the mode of inhibition of tetramic acids, the allosteric inhibitors of undecaprenyl pyrophosphate synthase.

Project description: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.

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Crystal structure of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase from the ESKAPE pathogen Acinetobacter baumannii.

Project description:The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the sixth step of the seven-step shikimate pathway. Chorismate, the product of the pathway, is a precursor for the biosynthesis of aromatic amino acids, siderophores and metabolites such as folate, ubiquinone and vitamin K. The shikimate pathway is present in bacteria, fungi, algae, plants and apicomplexan parasites, but is absent in humans. The EPSP synthase enzyme produces 5-enolpyruvylshikimate 3-phosphate and phosphate from phosphoenolpyruvate and shikimate 3-phosphate via a transferase reaction, and is the target of the herbicide glyphosate. The Acinetobacter baumannii gene encoding EPSP synthase, aroA, has previously been demonstrated to be essential during host infection for the growth and survival of this clinically important drug-resistant ESKAPE pathogen. Prephenate dehydrogenase is also encoded by the bifunctional A. baumannii aroA gene, but its activity is dependent upon EPSP synthase since it operates downstream of the shikimate pathway. As part of an effort to evaluate new antimicrobial targets, recombinant A. baumannii EPSP (AbEPSP) synthase, comprising residues Ala301-Gln756 of the aroA gene product, was overexpressed in Escherichia coli, purified and crystallized. The crystal structure, determined to 2.37 Å resolution, is described in the context of a potential antimicrobial target and in comparison to EPSP synthases that are resistant or sensitive to the herbicide glyphosate.

| S-EPMC4774876 | biostudies-literature

Structure-based inhibitors exhibit differential activities against Helicobacter pylori and Escherichia coli undecaprenyl pyrophosphate synthases.

Project description:Helicobacter pylori colonizes the human gastric epithelium and causes diseases such as gastritis, peptic ulcers, and stomach cancer. Undecaprenyl pyrophosphate synthase (UPPS), which catalyzes consecutive condensation reactions of farnesyl pyrophosphate with eight isopentenyl pyrophosphate to form lipid carrier for bacterial peptidoglycan biosynthesis, represents a potential target for developing new antibiotics. In this study, we solved the crystal structure of H. pylori UPPS and performed virtual screening of inhibitors from a library of 58,635 compounds. Two hits were found to exhibit differential activities against Helicobacter pylori and Escherichia coli UPPS, giving the possibility of developing antibiotics specially targeting pathogenic H. pylori without killing the intestinal E. coli.

| S-EPMC2276626 | biostudies-literature

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