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Exploration of GGTase-I substrate requirements. Part 2: Synthesis and biochemical analysis of novel saturated geranylgeranyl diphosphate analogs.

ABSTRACT: Protein prenylation is a type of post-translational modification that aids certain proteins in localizing to the plasma member where they activate cell signaling. To better understand the isoprenoid requirements and differences of FTase and GGTase-I, a series of saturated geranylgeranyl diphosphate analogs were synthesized and screened against both mammalian FTase and GGTase-I. Of our library of compounds, several analogs proved to be substrates of GGTase-I, with 11d having a krel=0.95 when compared to GGPP (krel=1.0).

SUBMITTER: Temple KJ

PROVIDER: S-EPMC4955811 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Exploration of GGTase-I substrate requirements. Part 2: Synthesis and biochemical analysis of novel saturated geranylgeranyl diphosphate analogs.

Temple Kayla J KJ Wright Elia N EN Fierke Carol A CA Gibbs Richard A RA

Bioorganic & medicinal chemistry letters 20160615 15

Protein prenylation is a type of post-translational modification that aids certain proteins in localizing to the plasma member where they activate cell signaling. To better understand the isoprenoid requirements and differences of FTase and GGTase-I, a series of saturated geranylgeranyl diphosphate analogs were synthesized and screened against both mammalian FTase and GGTase-I. Of our library of compounds, several analogs proved to be substrates of GGTase-I, with 11d having a krel=0.95 when comp ...[more]

PMID: 27342751

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Project description:BackgroundIsoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites.MethodsThe isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4-14C] isopentenyl diphosphate and three different allylic substrates: dimethylallyl diphosphate, geranyl diphosphate or farnesyl diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants KM and Vmax for each substrate were determined by Michaelis-Menten; also, inhibition assays were performed using risedronate.ResultsThe expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl diphosphate, as well as geranylgeranyl diphosphate, being therefore a bifunctional FPPS/geranylgeranyl diphosphate synthase (GGPPS) enzyme. The apparent KM values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68?±?5 ?M, 7.8?±?1.3 ?M and 2.06?±?0.4 ?M. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic parasites with a haemagglutinin-tagged version of FPPS. Also, the present data demonstrate that the recombinant protein is inhibited by risedronate.ConclusionsThe rPfFPPS is a bifunctional FPPS/GGPPS enzyme and the structure of products FOH and GGOH were confirmed mass spectrometry. Plasmodial FPPS represents a potential target for the rational design of chemotherapeutic agents to treat malaria.

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Exploration of GGTase-I substrate requirements. Part 2: Synthesis and biochemical analysis of novel saturated geranylgeranyl diphosphate analogs.

Publications

Exploration of GGTase-I substrate requirements. Part 2: Synthesis and biochemical analysis of novel saturated geranylgeranyl diphosphate analogs.

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