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Structural characterization of substrate and inhibitor binding to farnesyl pyrophosphate synthase from Pseudomonas aeruginosa.

Schmidberger JW, Schnell R, Schneider G - Acta Crystallogr. D Biol. Crystallogr. (2015)

Bottom Line: Two (in subunit A) and one (in subunit B) additional ibandronate molecules are bound in the active site.The structures of the fragment complexes show two molecules bound in a hydrophobic pocket adjacent to the active site.This allosteric pocket, which has previously only been described for FPPS from eukaryotic organisms, is thus also present in enzymes from pathogenic prokaryotes and might be utilized for the design of inhibitors of bacterial FPPS with a different chemical scaffold to the highly charged bisphosphonates, which are less likely to pass bacterial membranes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.

ABSTRACT
Locus PA4043 in the genome of Pseudomonas aeruginosa PAO1 has been annotated as coding for a farnesyl pyrophosphate synthase (FPPS). This open reading frame was cloned and expressed recombinantly in Escherichia coli. The dimeric enzyme shows farnesyl pyrophosphate synthase activity and is strongly inhibited by ibandronate and zoledronate, drugs that are presently in clinical use. The structures of the unliganded enzyme and complexes with the substrate geranyl diphosphate (GPP), the inhibitor ibandronate and two compounds obtained from a differential scanning fluorimetry-based screen of a fragment library were determined by X-ray crystallography to resolutions of better than 2.0 Å. The enzyme shows the typical α-helical fold of farnesyl pyrophosphate synthases. The substrate GPP binds in the S1 substrate site in an open conformation of the enzyme. In the enzyme-ibandronate complex three inhibitor molecules are bound in the active site of the enzyme. One inhibitor molecule occupies the allylic substrate site (S1) of each subunit, as observed in complexes of nitrogen-containing bisphosphonate inhibitors of farnesyl synthases from other species. Two (in subunit A) and one (in subunit B) additional ibandronate molecules are bound in the active site. The structures of the fragment complexes show two molecules bound in a hydrophobic pocket adjacent to the active site. This allosteric pocket, which has previously only been described for FPPS from eukaryotic organisms, is thus also present in enzymes from pathogenic prokaryotes and might be utilized for the design of inhibitors of bacterial FPPS with a different chemical scaffold to the highly charged bisphosphonates, which are less likely to pass bacterial membranes.

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The inhibitory effect of zoledronate (a) and ibandronate (b) on farnesyl pyrophosphate synthase from P. aeruginosa. The dose-response curves resulted in IC50 values of 15.2 nM and 2.7 µM, respectively.
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fig2: The inhibitory effect of zoledronate (a) and ibandronate (b) on farnesyl pyrophosphate synthase from P. aeruginosa. The dose-response curves resulted in IC50 values of 15.2 nM and 2.7 µM, respectively.

Mentions: This assignment is further strengthened by the influence of well known FPPS inhibitors that are in clinical use: ibandronate and zoledronate. Both compounds were shown to inhibit PaFPPS in a concentration-dependent manner, with an IC50 value of 2.7 ± 0.4 µM for ibandronate, while zoledronate is a considerably stronger inhibitor with an IC50 value of 15.2 ± 0.7 nM (Fig. 2 ▶). This parallels the relative effects of these drugs on FPPS from human and T. brucei, with zoledronate being one of the strongest N-BP inhibitors (Russell et al., 2008 ▶). Zoledronate inhibits the human enzyme with an IC50 of 4.1 nM (Kavanagh et al., 2006 ▶) and the trypanosomal enzyme with an IC50 of 400 nM (Yin et al., 2006 ▶). The corresponding IC50 values for ibandronate are 20 nM (Dunford et al., 2001 ▶) and 3.1 µM (Yin et al., 2006 ▶), respectively.


Structural characterization of substrate and inhibitor binding to farnesyl pyrophosphate synthase from Pseudomonas aeruginosa.

Schmidberger JW, Schnell R, Schneider G - Acta Crystallogr. D Biol. Crystallogr. (2015)

The inhibitory effect of zoledronate (a) and ibandronate (b) on farnesyl pyrophosphate synthase from P. aeruginosa. The dose-response curves resulted in IC50 values of 15.2 nM and 2.7 µM, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356374&req=5

fig2: The inhibitory effect of zoledronate (a) and ibandronate (b) on farnesyl pyrophosphate synthase from P. aeruginosa. The dose-response curves resulted in IC50 values of 15.2 nM and 2.7 µM, respectively.
Mentions: This assignment is further strengthened by the influence of well known FPPS inhibitors that are in clinical use: ibandronate and zoledronate. Both compounds were shown to inhibit PaFPPS in a concentration-dependent manner, with an IC50 value of 2.7 ± 0.4 µM for ibandronate, while zoledronate is a considerably stronger inhibitor with an IC50 value of 15.2 ± 0.7 nM (Fig. 2 ▶). This parallels the relative effects of these drugs on FPPS from human and T. brucei, with zoledronate being one of the strongest N-BP inhibitors (Russell et al., 2008 ▶). Zoledronate inhibits the human enzyme with an IC50 of 4.1 nM (Kavanagh et al., 2006 ▶) and the trypanosomal enzyme with an IC50 of 400 nM (Yin et al., 2006 ▶). The corresponding IC50 values for ibandronate are 20 nM (Dunford et al., 2001 ▶) and 3.1 µM (Yin et al., 2006 ▶), respectively.

Bottom Line: Two (in subunit A) and one (in subunit B) additional ibandronate molecules are bound in the active site.The structures of the fragment complexes show two molecules bound in a hydrophobic pocket adjacent to the active site.This allosteric pocket, which has previously only been described for FPPS from eukaryotic organisms, is thus also present in enzymes from pathogenic prokaryotes and might be utilized for the design of inhibitors of bacterial FPPS with a different chemical scaffold to the highly charged bisphosphonates, which are less likely to pass bacterial membranes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.

ABSTRACT
Locus PA4043 in the genome of Pseudomonas aeruginosa PAO1 has been annotated as coding for a farnesyl pyrophosphate synthase (FPPS). This open reading frame was cloned and expressed recombinantly in Escherichia coli. The dimeric enzyme shows farnesyl pyrophosphate synthase activity and is strongly inhibited by ibandronate and zoledronate, drugs that are presently in clinical use. The structures of the unliganded enzyme and complexes with the substrate geranyl diphosphate (GPP), the inhibitor ibandronate and two compounds obtained from a differential scanning fluorimetry-based screen of a fragment library were determined by X-ray crystallography to resolutions of better than 2.0 Å. The enzyme shows the typical α-helical fold of farnesyl pyrophosphate synthases. The substrate GPP binds in the S1 substrate site in an open conformation of the enzyme. In the enzyme-ibandronate complex three inhibitor molecules are bound in the active site of the enzyme. One inhibitor molecule occupies the allylic substrate site (S1) of each subunit, as observed in complexes of nitrogen-containing bisphosphonate inhibitors of farnesyl synthases from other species. Two (in subunit A) and one (in subunit B) additional ibandronate molecules are bound in the active site. The structures of the fragment complexes show two molecules bound in a hydrophobic pocket adjacent to the active site. This allosteric pocket, which has previously only been described for FPPS from eukaryotic organisms, is thus also present in enzymes from pathogenic prokaryotes and might be utilized for the design of inhibitors of bacterial FPPS with a different chemical scaffold to the highly charged bisphosphonates, which are less likely to pass bacterial membranes.

Show MeSH
Related in: MedlinePlus