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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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(a) Chemical structures of bisphosphonates. Nitrogen-containing bisphosphonates such as zoledronate and ibandronate have a hydroxyl group in the R1 position and nitrogen-containing R2 side chains. (b) Farnesyl pyrophosphate synthase-catalyzed condensation reactions. Isopentenyl diphosphate (IPP) first reacts with dimethylallyl diphosphate (DMAPP) to form geranyl diphosphate (GPP), releasing pyrophosphate (PPi). GPP then reacts with IPP to form farnesyl pyrophosphate (FPP) and free PPi.
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fig1: (a) Chemical structures of bisphosphonates. Nitrogen-containing bisphosphonates such as zoledronate and ibandronate have a hydroxyl group in the R1 position and nitrogen-containing R2 side chains. (b) Farnesyl pyrophosphate synthase-catalyzed condensation reactions. Isopentenyl diphosphate (IPP) first reacts with dimethylallyl diphosphate (DMAPP) to form geranyl diphosphate (GPP), releasing pyrophosphate (PPi). GPP then reacts with IPP to form farnesyl pyrophosphate (FPP) and free PPi.

Mentions: Farnesyl pyrophosphate synthase (FPPS; EC 2.5.1.10) is a promising antibiotic drug target as it represents the point of convergence of these two pathways (Dhar et al., 2013 ▶). FPPS has been listed as indispensible for the survival for a number of pathogenic bacteria (Luo et al., 2014 ▶). The enzyme belongs to a large group of prenyltransferases responsible for the principal bond-forming reactions of isoprenoid biosynthesis (Poulter et al., 1978 ▶; Song & Poulter, 1994 ▶). The mevalonate and nonmevalonate pathways both produce isopentenyl pyrophosphate (IPP), which along with its isomer dimethyl­allyl diphosphate (DMAPP) is converted through a ‘head-to-tail’ condensation reaction to form geranyl diphosphate (GPP) by FPPS (Fig. 1 ▶). While prenyltransferases do exhibit some range in selectivity of product length, FPPS tends not to synthesize a prenyl chain longer than the 15-carbon isoprenoid chain of farnesyl pyrophosphate (FPP; Tarshis et al., 1996 ▶; Dhar et al., 2013 ▶). FPP is involved in protein prenylation and in the formation of triterpenes, both of which are important for cell survival (Oldfield, 2010 ▶).


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)

(a) Chemical structures of bisphosphonates. Nitrogen-containing bisphosphonates such as zoledronate and ibandronate have a hydroxyl group in the R1 position and nitrogen-containing R2 side chains. (b) Farnesyl pyrophosphate synthase-catalyzed condensation reactions. Isopentenyl diphosphate (IPP) first reacts with dimethylallyl diphosphate (DMAPP) to form geranyl diphosphate (GPP), releasing pyrophosphate (PPi). GPP then reacts with IPP to form farnesyl pyrophosphate (FPP) and free PPi.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: (a) Chemical structures of bisphosphonates. Nitrogen-containing bisphosphonates such as zoledronate and ibandronate have a hydroxyl group in the R1 position and nitrogen-containing R2 side chains. (b) Farnesyl pyrophosphate synthase-catalyzed condensation reactions. Isopentenyl diphosphate (IPP) first reacts with dimethylallyl diphosphate (DMAPP) to form geranyl diphosphate (GPP), releasing pyrophosphate (PPi). GPP then reacts with IPP to form farnesyl pyrophosphate (FPP) and free PPi.
Mentions: Farnesyl pyrophosphate synthase (FPPS; EC 2.5.1.10) is a promising antibiotic drug target as it represents the point of convergence of these two pathways (Dhar et al., 2013 ▶). FPPS has been listed as indispensible for the survival for a number of pathogenic bacteria (Luo et al., 2014 ▶). The enzyme belongs to a large group of prenyltransferases responsible for the principal bond-forming reactions of isoprenoid biosynthesis (Poulter et al., 1978 ▶; Song & Poulter, 1994 ▶). The mevalonate and nonmevalonate pathways both produce isopentenyl pyrophosphate (IPP), which along with its isomer dimethyl­allyl diphosphate (DMAPP) is converted through a ‘head-to-tail’ condensation reaction to form geranyl diphosphate (GPP) by FPPS (Fig. 1 ▶). While prenyltransferases do exhibit some range in selectivity of product length, FPPS tends not to synthesize a prenyl chain longer than the 15-carbon isoprenoid chain of farnesyl pyrophosphate (FPP; Tarshis et al., 1996 ▶; Dhar et al., 2013 ▶). FPP is involved in protein prenylation and in the formation of triterpenes, both of which are important for cell survival (Oldfield, 2010 ▶).

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