Plasmodium falciparum spermidine synthase with dcAdoMet

Target ID:

PF11_0301

Deposition Date:

Tuesday 8th May 2007

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

protein-ligand

Download Coordinates:

2PT9

Authors:

Dufe, V.T., Qui, W, Muller, I, Hui, R, Walter, R, Al-Karadaghi, S

Site:

Toronto

2PT9

tabs

Structure Details

Polyamines are essential for normal cell proliferation and cell differentiation of prokaryotes and eukaryotes.
Common polyamines such as putrescine, spermidine and spermine are distributed ubiquitously in cells and tissues at relatively high concentration.

Enzymes involved in polyamines pathway mainly include ornithine decarboxylase (ODC), S-Adenosylmethionine decarboxylase (AdoMetDC), spermidine synthase (SRM), spermine synthase (SMS), spermidine/spermine N1-acetyltransferase (SSAT) and FAD-dependent polyamine oxidase (PAO).
The polyamines are suggested to be more critical for rapidly growing and replicating P. falciparum than for normal human cells.
It has been reported that some of these polyamine enzymes exhibit features that differ significantly between the parasites and the human host.
Thus, the exploitation of such differences may lead to the design of new inhibitors that will selectively kill the parasites while exerting minimal effects on the parasite-infected host.

Spermidine synthase is an aminopropyltransferase that transfers of an aminopropyl moiety of dcAdoMet onto putrescine, leading to the formation of spermidine and 5′-methylthioadenosine (MTA).
In our recently solved crystal structure of Pf-SRM, one of the products, MTA can be clearly seen in the active binding site.

Pf-SRM is a dimer in the crystal and in solution (confirmed by size exclusion chromatography).
The overall structure, as well as the substrate binding pocket is similar to that of
human SRM,
also a structure solved by the SGC. Superimposition of the two structures is presented to the right.
The Pf-SRM monomer consists of two domains: a small N-terminal domain and a large catalytic C-terminal domain (also known as SAM-dependent methyltransferase domain).
A PART (putrescine aminopropyltransferase) family signature V-(LA)-(LIV)(2)-G-G-G-X-G-X(2)-(LIV)-X-E (where X is any amino acid) sequence is also found in Pf-SRM. Some catalytic residues are located in the loop β10-αE (residue 158-169), which are disordered in two out of three molecules in the asymmetric unit.

It is of interest to note that, Pf-SRM protein was over-expressed from synthetic DNA since the gene encoding Pf-SRM consists of four exons and three introns on P. falciparum genomic DNA (www.plasmodb.org).

Materials and Methods

Pf-SRM + dcAdoMet

PDB:2PT9

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:PF11_0301
Entry Clone Source:Codon optimized synthetic DNA
SGC Clone Accession:
Tag:N-terminal: His6-tag with integrated TEV protease site: mgsshhhhhhssgrenlyfq*g
Host:E. coli BL21-(DE3)-R3

Construct

Prelude:
Sequence:gSKKWFSEFSIMWPGQAFSLKIKKILYETKSKYQNVLVFESTTYGKVLVLDGVIQLTEKDEFAYHEMMTHVPMTVSKEPKNVLVVGGGDGGIIRELCKYKSVENIDICEIDETVIEVSKIYFKNISCGYEDKRVNVFIEDASKFLENVTNTYDVIIVDSSDPIGPAETLFNQNFYEKIYNALKPNGYCVAQCESLWIHVGTIKNMIGYAKKLFKKVEYANISIPTYPCGCIGILCCSKTDTGLTKPNKKLESKEFADLKYYNYENHSAAFKLPAFLLKEIENI
Vector:p15-Tev-LIC

Growth

Medium:TB
Antibiotics:
Procedure:Pf-SRM was expressed in E. coli BL21-(DE3)-Rosetta Oxford in Terrific Broth (TB) in the presence of ampicillin/chloramphenicol (100 μg/mL and 34 μg/mL respectively). A single colony was inoculated into 100mL of LB with of ampicillin/chloramphenicol (100 μg/mL and 34 μg/mL respectively) in a 250 mL baffled flask and incubated with shaking at 250 rpm overnight at 37 ºC. The culture was transferred into 1.8 L of TB with ampicillin/chloramphenicol (100 μg/mL and 34 μg/mL respectively) and 0.3 mL of antifoam (Sigma) in a 2 L bottle and cultured using the LEX system to an OD600 of 4.5, cooled to 15 ºC, and induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15 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). The DE52 column was further washed with 20 mL of Binding Buffer.
The lysate was subsequently loaded onto a 1.0-2.5 mL Ni-NTA (Qiagen) column (pre-equilibrated with Binding Buffer) at approximately 1-1.5 mL/min. The Ni-NTA column was then washed with 200 mL of Wash Buffer at 2-2.5 mL/min. After washing, the protein was eluted with 15 mL of Elution Buffer.EDTA was immediately added to the elution fraction to 1 mM; and DTT was added to 5 mM after approximately 15 more minutes. The eluted Pf-SRM was applied to a Sephadex S200 26/60 gel filtration column pre-equilibrated with 10 mM HEPES, pH 7.5 and 500 mM NaCl. The fractions corresponding to the eluted protein peak were collected.
The His-tag was cleaved with TEV protease overnight at 4 ºC in the presence of 1mM DTT. The cleaved sample was applied to a 1mL Ni-NTA column pre-equilibrated with Binding buffer. The flow-through was collected; and the column was rinsed with an additional 5 mL of Binding Buffer. These fractions were pooled and concentrated using a 15 mL Amicon Ultra centrifugal filter device (Millipore). Using the same device, Binding Buffer was exchanged to Crystal Buffer by adding 15 mL Crystal Buffer twice to the portion of protein solution that was concentrated to around 0.5 mL.Finally, the protein was concentrated and flash frozen and stored at -80 ºC.

Extraction

Procedure
The culture was harvested by centrifugation. Pellets from 4 L of culture 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 °C 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 16000 psi; and the cell lysate was centrifuged using a Beckman JA-25.50 rotor at 24000 rpms (~75000 x g) for 20 minutes at 10 ºC.
Concentration:14.2 mg/mL for Pf-SRM (construct ID: PF11_0301:s:S40-I321:E7, without histag).
Ligand
MassSpec:
Crystallization:Purified Pf-SRM was crystallized using the sitting drop vapor diffusion method at 18 ºC in 25% PEG3350, 0.1 M Ammonium Sulphate and 0.1 M Bis-Tris pH 5.5 and 2.5 mM dcAdoMet.
NMR Spectroscopy:
Data Collection:
Data Processing: