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Structure Details

Plasmodium vivax farnesyl pyrophosphate synthase

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PDB Code 2IHI Target Class Malaria

Target Pv-PF11_0295
Alias n/a
Disease Area/Function parasitic disease
Date Deposited 2006-09-26
Authors A.DONG, J.DUNFORD, J.LEW, A.K.WERNIMONT, H.REN, Y.ZHAO, I.KOEIERADZKI, U.OPPERMAN, M.SUNDSTROM, J.WEIGELT, A.M.EDWARDS, C.H.ARROWSMITH, A.BOCHKAREV, R.HUI, J.D.ARTZ, STRUCTURALGENOMICS CONSORTIUM (SGC)
Related Structure 3CC9

Struc Details Tabs

Structure Details
Isoprenoids such as sterols and ubiquinones are a diverse and universal family of natural products. Their biosynthesis typically involves condensation of different numbers of isopentenyl diphosphate units (IPP).1 In mammals, IPP is produced by means of the mevalonate pathway with eponymous mevalonic acid as the starting metabolite. Simpler organisms, including eubacteria, algae and some plants, undergo an alternative non-mevalonate mechanism involving conversion of glyceraldehyde 3-phosphate (G3P) and pyruvate to 1-deoxy-D-xylulose 5-phosphate (DOXP) and subsequently to 2-C-methyl-D-erythritol 4-phosphate (MEP), giving rise to the DOXP pathway or the MEP pathway.

With studies uncovering the ineffectiveness of inhibitors of the mevalonate pathway, identification of enzymes such as DOXP synthase and DOXP reductoisomerase, as well as effectiveness of fosmidomycin in curing malaria in mice,2 it has been established that Plasmodium parasites produce IPP by means of the MEP pathway. Specifically, it has been shown that this pathway takes place inside the apicoplast organelle in both Plasmodium and Toxoplasma parasites.

Once IPP and its isomer dimethylallyl diphosphate (DMAPP) are produced, they are converted to first geranyl pyrophosphate (GPP) and finally the isoprenoid precursor farnesyl pyrophosphate (FPP) by the catalytic action of farnesyl pyrophosphate synthase (FPPS). This is a well characterized enzyme which is also a validated drug target for osteoporosis in humans and considered a potential drug target in protozoan parasites. Specifically, nitrogen-containing bisphosphonates, which are non-hydrolyzable pyrophosphate analogues and proven FPPS inhibitors (with some compounds currently available as drugs against osteoporosis), have been found to have nanomolar to low micro-molar activity in vitro against Trypanosoma, Leishmania, Toxoplasma and Plasmodium parasites.3

We have solved the structure of a Plasmodium vivax enzyme who is homologous by sequence to human FPPS and GGPPS. Enzymatic characterization showed that this enzyme produced exclusively GGPP but was inhibited at mid-nanomolar concentrations by N-BPs, which is uncommon for a geranylgeranyl pyrophosphate synthase. This structure will aid in further elucidation of the isoprenoid pathway in Plasmodium parasites and provide the basis for structure guided optimization of FPPS inhibitors as anti-malarials.

See also

  • SGC's human FPPS structure with in complex with risedronate and zoledronate
  • SGC's Cryptosporidium parvum structure of polyprenyl pyrophosphate synthase in complex with zoledronate

References

  1. Rohmer, M. (2003) "Mevalonate-independent methylerythritol phosphate pathway for isoprenoid biosynthesis. Elucidation and distribution." Pure Appl. Chem., Vol. 75, Nos. 2–3, pp. 375–387.
  2. Jomaa, H.,Wiesner, J., Sanderbrand, S., Altincicek, B., Weidemeyer, C., Hintz, M., Tu¬rbachova, I., Eberl, M., Zeidler, J., Lichtenthaler, H., Soldati, D. and Beck, E. (1999) "Inhibitors of the Nonmevalonate Pathway of Isoprenoid Biosynthesis as Antimalarial Drugs." Science, Vol. 285, No 3, pp. 1573-6.
  3. Martin, M.B., Grimley, J.S., Lewis, J.C., Heath, III, H.T., Bailey, B.N., Kendrick, H., Yardley, V., Caldera, A., Renee Lira, R., Urbina, J.A., Moreno, S.N.J., Docampo, R., Croft, S.L. and Oldfield, E. (2001). "Bisphosphonates Inhibit the Growth of Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Plasmodium falciparum: A Potential Route to Chemotherapy." J. Med. Chem.
Materials & Methods
StructurePv-FDPS: Plasmodium vivax farnesyl diphosphate synthase
PDB Code2IHI
Entry clone accession Pv092040
Entry clone source Plasmodium vivax Salvador I genomic DNA
SGC clone accession Pv-PF11_0295; plate MAC01Q:A12
Tag N-terminal: His6-tag with integrated TEV protease site: mhhhhhhssgrenlyfq*g
Construct sequencemgsshhhhhhssgrenlyfqgMKETNSEEADSGLAFFRNMYDKYRDAFLSHLNEYSLEEEIKEHISKYYKLLFDYNCLGG
KNNRGILVILIYEYVKNRDINSSEWEKAACLAWCIEILQAAFLVADDIMDKGEMRRNKYCWYLLKDVETKNAVNDVLLLY
NSIYKLIEIYLRNESCYVDVIATFRDATLKTIIGQHLDTNIFSDKYSDAHREIDVNNINVPEQPVIDINMINFGVYKNIV
IHKTAYYSFFLPIVCGMLLAGIAVDNLIYKKIEDISMLMGEYFQIHDDYLDIFGDSTKTGKVGSDIQNNKLTWPLIKTFE
LCSEPDKIKIVKNYGKNNLACVKVIDSLYEQYKIRKHYESYEKAQKAKILSAINELHHEGIEYVLKYLLEILFTGV
Vector p15-tev-lic
Expression host E. coli BL21-(DE3)-R3-pRARE2
Growth method A single colony was inoculated into 10 mL of LB with of Antibiotics and incubated with shaking at 250 rpm overnight at 37 ºC. The culture was transferred into 50 mL of TB with Antibiotics in a 250 mL shaking flask and incubated at 37 ºC for 3 hours. The culture was then transferred into 1.8 L of above-specified growth medium with Antibiotics and 0.3 mL of antifoam (Sigma) in a 2 L bottle and cultured using the LEX system to an OD600 of ~5, cooled to 15 ºC and induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15 ºC.
Extraction buffersBinding Buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM imidazole, and 5 % glycerol
Extraction procedure Cells were resuspended to approximately 40 mL/L of cell culture in Binding Buffer with the addition of protease inhibitors (1 mM benzamidine and 1 mM phenylmethyl sulfonyl fluoride (PMSF)). Resuspended pellets stored at -80 oC were thawed overnight at 4 ºC on the day before purification. Prior to mechanical lysis, each pellet from 1 L of culture was pretreated with 0.5% CHAPS and 500 units of benzonase for 40 minutes at room temperature. Cells were mechanically lysed with a microfluidizer (Microfluidizer Processor, M-110EH) at approximately 18000 psi; and the cell lysate was centrifuged using a Beckman JA-25.50 rotor at ~75000 x g (24000 rpms) for 20 minutes at 10 oC.
Purification procedure The cleared lysate was loaded onto a column prepacked with 10 g DE52 (Whatman) anion exchange resin (previously activated with 2.5 M NaCl and equilibrated with Binding Buffer) and subsequently onto a 1.0 – 2.5 mL Ni-NTA (Qiagen) column pre-equilibrated with Binding Buffer at approximately 1 – 1.5 mL/min. After the lysate was loaded, the DE52 was further washed with 20 mL of Binding Buffer. Each Ni-NTA column was then washed with 200 mL of Wash Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 30 mM imidazole, and 5 % glycerol) at 2 – 2.5 mL/min. After washing, the protein was eluted with 15 mL of Elution Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 250 mM imidazole, and 5 % glycerol). EDTA was immediately added to the elution fraction to 1 mM; and DTT was added to 1 – 5 mM after approximately 15 more minutes. The sample was loaded onto a Sephadex S200 26/60 gel filtration column pre-equilibrated with 10 mM HEPES, pH 7.5 and 500 mM NaCl. The collected fractions corresponding to the correct eluted protein peak were concentrated using a 15 mL Amicon Ultra centrifugal filter device (Millipore). The protein sample identity and purity were evalulated by mass spectroscopy and SDS-PAGE gel. The concentrated sample was stored at 4 oC.
Protein stock concentration 16.5 mg/mL for Pv-FDPS with His6-tag.
Crystallization The native protein was crystallized by means by hanging drop vapor diffusion in a VDXm plate. The plate was set with 1.5 μL protein and 1.5 μL buffer in each drop, and 350 μL reservoir volume per well. Crystals grew overnight in 22% Peg 3350, 200 mM Li2SO4, 100 mM Tris, pH 8.5 at 18 ºC.
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