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Py-ACPS: Plasmodium yoelii holo-(acyl-carrier protein) synthase, putative

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PDB Code 2BDD Target Class Malaria

Target PY06285
Alias n/a
Disease Area/Function parasitic disease
Date Deposited 2005-10-20
Authors A.DONG, M.MELONE, Y.ZHAO, J.LEW, I.KOEIERADZKI, Z.ALAM, G.WASNEY, M.VEDADI, A.M.EDWARDS, C.H.ARROWSMITH, J.WEIGELT, M.SUNDSTROM, A.BOCHKAREV, R.HUI, M.AMANI, STRUCTURAL GENOMICS CONSORTIUM(SGC)
Related Structure 2QG8

Struc Details Tabs

Structure Details
Essential to all organisms, fatty acids make up all biomembranes and supply energy. Animals typically undergo Type I fatty acid biosynthesis (FAS), in which large multifunctional enzymes catalyze all fatty acid chain elongation reactions. In contrast, plants and bacteria rely on the Type II system, where each elongation reaction employs a distinct enzyme. This difference makes Type II FAS enzymes opportune targets for antibiotic drug discovery.

Protozoans, specifically apicomplexans, are mixed in their FAS mechanisms. Malaria and Cryptosporidium parasites (C. parvum and C. hominis) respectively employ Type II and I routes, while both mechanisms co-exist in Toxoplasma gondii.

Type II features post-translational modification of acyl carrier protein (ACP) into holo-ACP - transfer of a 4′-phosphopantetheine (PPT) group from coenzyme A (CoA) to a conserved serine residue by means of catalytic action of holo-ACP synthase (holo-ACPS), a type of phosphopantetheinyl transferase. During fatty acid synthesis, the acyl intermediates are bound through a thioester linkage to this PPT moiety.
We have solved the structure of the C-terminal catalytic domain (H361-V521), of holo-ACP synthase from Plasmodium yoelii, namely PY06285, that is 63% identical to the Plasmodium falciparum ortholog PFD0980w. Py-holo-ACPS features a homo-trimer with each monomer contributing three anti-parallel beta sheets to form a barrel-like active site. Although many FAS enzymes, including ACP, in Plasmodium genomes are targeted for the apicoplast, this is not the case for holo-ACPS, suggesting that ACP is modified before entering the organelle.

See other malaria related structures.

References

1. Cai, X., Herschap, D. and Zhu. G. Functional Characterization of an Evolutionarily Distinct Phosphopantetheinyl Transferase in the Apicomplexan Cryptosporidium parvum. Eukaryotic Cell, July 2005, p. 1211-1220, Vol. 4, No. 7.

2. Norman C. Watersa, Karen M. Kopydlowskia, Tadeusz Guszczynskib, Lan Weia, Patrice Sellersa, Jill T. Ferlana, Patricia J. Leea, Zhiyu Lia, Cassandra L. Woodarda, Shamira Shallomc, Malcolm J. Gardnerc and Sean T. Prigge. Functional characterization of the acyl carrier protein (PfACP) and beta-ketoacyl ACP synthase III (PfKASIII) from Plasmodium falciparum. Molecular and Biomedical Parasitology, 28 Aug 2002. Vol 123, Issue2

3. Chirgadze NY, Briggs SL, McAllister KA, Fischl AS, Zhao G. Crystal structure of Streptococcus pneumoniae acyl carrier protein synthase: an essential enzyme in bacterial fatty acid biosynthesis. EMBO J. 2000 Oct 16;19(20):5281-7.

Materials & Methods
StructurePy-holo-ACPS: Plasmodium yoelii holo acyl carrier protein synthase (PY06285)
PDB Code2BDD
Entry clone accession PY06285
Entry clone source Plasmodium yoelii 17XNL genomic DNA
SGC clone accession PY06285:H361-V521; plate MAC00I:B7
Tag N-terminal: N-terminal His-tag with integrated TEV protease site (*): MGSSHHHHHHSSGRENLYFQ*G
Construct sequencemgsshhhhhhssgrenlyfqgHHIIGIGTDILCVNRIYKILEKNINFIKKVLNPFELAEFETQKKKLNEKINKSNELKKL
AIYVSKKFAAKEAILKSMGRGLSSISKYGLSMNDIEIKNDKYGKPHVYLYGKAKKVAYEMGIVKIFLSISDEKIINSQTN
NISSNFPTFIIQAQALAVGSNV
Vector p15TV-L
Expression host E. coli BL21-(DE3)-CodonPlus-RIL from Stratagene
Growth medium Terrific Broth (TB)
Antibiotics 50 microG/mL kanamycin and 25 microG/mL chloramphenicol
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 2L bottle and cultured using the LEX system to an OD600 of 2.5, cooled to 15 ºC and induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15 ºC.
Extraction buffersBinding buffer: 10 mM HEPES pH 7.5, 0.5 M NaCl, 5% glycerol, 5 mM imidazole
Extraction procedure Cells were resuspended to approximately 40 mL/L of cell culture in Binding Buffer with protease inhibitor (1 mM benzamidine-HCl 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 18,000 psi. The cell lysate was centrifuged at ~75,000 x g for 20 minutes at 10 ºC.
Purification buffersWash buffer: 10mM HEPES pH 7.5, 0.5 M NaCl, 5% glycerol, 30 mM imidazole
Elution buffer: 10mM HEPES, pH 7.5, 0.5 M NaCl, 5% glycerol, 250 mM imidazole
Gel filtration bffer: 10 mM HEPES, pH 7.5 and 500 mM NaCl
Crystal buffer: 10 mM HEPES, pH 7.5 and 500 mM NaCl
Purification procedure The cleared cell lysate was loaded onto a DE52 (Whatman) column packed with 10 g of resin (previously activated with 3 M NaCl and equilibrated with Binding Buffer), and subsequently onto a 2.5 mL Ni-NTA column at approximately 1.5 mL/min. When all the lysate was loaded, 20 mL of Binding Buffer was added to the DE52 column. Then the Ni-NTA column was washed with 200 mL of Wash Buffer at 2–2.5 mL/min. After washing, the protein was eluted from the Ni-NTA column with 15 mL of Elution Buffer. EDTA was added immediately to 1 mM. DTT was then added to 1 mM 15 minutes later.

The protein was dialysed overnight in a dialysis cassette (Pierce) in Crystal Buffer. The following day, it was concentrated using a 15 mL Amicon Ultra centrifugal filter device Millipore (5 kD cutoff).Protein concentration was estimated by means of absorbance at OD280. Aliquots of the purified protein were flash frozen in N2(l) and stored at -80°C.

Protein stock concentration 12.5 mg/mL
Crystallization The protein was crystallized by means of hanging drop vapor diffusion in Linbro plate. The plate was set with 1.5 μL protein and 1.5 μL buffer in each drop, and 500 μL reservoir volume per well. Crystals grew overnight in 16% PEG8K, 0.2M Mg Acetate and 0.1M Na Cacodylate at 18 ºC.
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