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

Target ID:

PY06285

Deposition Date:

Thursday 20th October 2005

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

2BDD

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)

Site:

Toronto

ICM Datapack:

ICM Datapack

2BDD

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.

Materials and Methods

Py-holo-ACPS: Plasmodium yoelii holo acyl carrier protein synthase (PY06285)

PDB:2BDD

Revision

Revision Type:created
Revised by:created
Revision Date:created
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
Host:E. coli BL21-(DE3)-CodonPlus-RIL from Stratagene

Construct

Prelude:
Sequence:mgsshhhhhhssgrenlyfqgHHIIGIGTDILCVNRIYKILEKNINFIKKVLNPFELAEFETQKKKLNEKINKSNELKKLAIYVSKKFAAKEAILKSMGRGLSSISKYGLSMNDIEIKNDKYGKPHVYLYGKAKKVAYEMGIVKIFLSISDEKIINSQTNNISSNFPTFIIQAQALAVGSNV
Vector:p15TV-L

Growth

Medium:Terrific Broth (TB)
Antibiotics:50 microG/mL kanamycin and 25 microG/mL chloramphenicol
Procedure:A single colony was inoculated into 10 mL of LB with of Antibiotics and incubated with shaking at 250 rpm overnight at 37 degC. The culture was transferred into 50 mL of TB with Antibiotics in a 250 mL shaking flask and incubated at 37 degC 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 degC and induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15 degC.

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 2Â2.5 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.

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 degC were thawed overnight at 4 degC 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 degC.
Concentration:12.5 mg/mL
Ligand
MassSpec:
Crystallization:The protein was crystallized by means of hanging drop vapor diffusion in Linbro plate. The plate was set with 1.5 microL protein and 1.5 microL buffer in each drop, and 500 microL reservoir volume per well. Crystals grew overnight in 16% PEG8K, 0.2M Mg Acetate and 0.1M Na Cacodylate at 18 degC.
NMR Spectroscopy:
Data Collection:
Data Processing:

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.