Abstract
We present here a study of a eukaryotic trans-prenylsynthase from the malaria pathogen Plasmodium vivax. Based on the results of biochemical assays and contrary to previous indications, this enzyme catalyzes the production of geranylgeranyl pyrophosphate (GGPP) rather than farnesyl pyrophosphate (FPP). Structural analysis shows that the product length is constrained by a hydrophobic cavity formed primarily by a set of residues from the same subunit as the product as well as at least one other from the dimeric partner. Furthermore, Plasmodium GGPP synthase (GGPPS) can bind nitrogen-containing bisphosphonates (N-BPs) strongly with the energetically favorable cooperation of three Mg2+, resulting in inhibition by this class of compounds at IC50 concentrations below 100 nm. In contrast, human and yeast GGPPSs do not accommodate a third magnesium atom in the same manner, resulting in their insusceptibility to N-BPs. This differentiation is in part attributable to a deviation in a conserved motif known as the second aspartate-rich motif: whereas the aspartates at the start and end of the five-residue motif in FFPP synthases and P. vivax GGPPSs both participate in the coordination of the third Mg2+, an asparagine is featured as the last residue in human and yeast GGPPSs, resulting in a different manner of interaction with nitrogen-containing ligands.
Highlights
Precursors to steroids and sterols, they are utilized by prokaryotes and eukaryotes alike to modify proteins, such as G proteins, kinases, and phosphatases, and facilitate their localization to membranes
The T. gondii farnesyl pyrophosphate synthase (FPPS), sensitive to inhibition by Nitrogen-containing bisphosphonates (N-BPs), produces farnesyl pyrophosphate (FPP) along with a low level of geranylgeranyl pyrophosphate (GGPP) when dosed with a high concentration of FPP [16]; this bifunctional behavior is upstaged by a C. parvum nonspecific polyprenyl pyrophosphate synthase (CpNPPPS) that can synthesize a great range of products from C20 to C40 and can be inhibited by N-BPs at IC50 concentrations below 100 nM [12]
The two Plasmodium orthologs are Ͼ70% identical to each other and approximately 30% identical to Trypanosoma and Toxoplasma FPPS as well as CpNPPPS, but less similar in sequence to geranylgeranyl pyrophosphate synthase (GGPPS) from E. histolytica
Summary
Reagents—All inhibitors used in this study, namely RIS, ZOL, and ibandronate (IBAN) were provided by Procter & Gamble Pharmaceuticals (Cincinnati, OH). Crystallization, Data Collection, Structure Solution, and Refinement—PvGGPPS was crystallized in its apo form by mixing 1.5 l of protein (at 16.5 mg/ml concentration in a buffer of 10 mM HEPES, pH 7.5, 500 mM NaCl) with 1.5 l of reservoir solution consisting of 22% PEG 3350, 200 mM Li2SO4, 100 mM Tris, pH 8.5, in a hanging drop vapor diffusion setup with Ͼ350 l of reservoir solution at 18 °C in VDXm plates (Hampton Research). For GGPP-added crystals, 1.5 l of protein (at 12.1 mg/ml in 10 mM HEPES, pH 7.5, 500 mM NaCl containing 1 mM GGPP and 2 mM MgCl2) was mixed with 1.5 l of reservoir solution consisting of 25% PEG 3350, 200 mM (NH4)2SO4, 100 mM Tris, pH 8.5, and incubated in hanging drops over 350 l of reservoir solution at 18 °C. The native structure solution was obtained by molecular replacement using the program PHASER and Protein Data Bank (PDB) ID code 1UBV (Gallus gallus FPPS) as a model. The final models have good geometry and no outliers in the Ramachandran analysis
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