Plasmodium berghei orotidine monophosphate decarboxylase, putative

Target ID:

PF10_0225

Deposition Date:

Thursday 15th December 2005

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

2FDS

Authors:

W.QIU,A.DONG,G.WASNEY,M.VEDADI,J.LEW,I.KOZIERADSKI,Z.ALAM,M.MELONE,J.WEIGELT,M.SUNDSTROM,A.EDWARDS,C.ARROWSMITH,R.HUI,M.GAO,A.BOCHKAREV,J.D.ARTZ,STRUCTURAL GENOMICS CONSORTIUM(SGC)

Site:

Toronto

2FDS

tabs

Structure Details

In Plasmodium parasites, pyrimidine nucleotides are synthesized de novo
via a six-step pathway1.
One of the steps involves addition of a ribosyl phosphate group
to orotic acid by orotate phosphoribosyltransferase (OPRT),
resulting to orotidine 5'-monophosphate (OMP).
Subsequently, orotidine 5'-monophosphate decarboxylase (OMPDC)
catalyses the removal of the carboxyl group from OMP to produce,
uridine monophosphate (UMP),
from which all pyrimidine nucleotides can be derived.
This decarboxylation step is accelerated by a factor of 1017
without the benefit of a catalytic cofactor by OMPDC,
making it the most efficient of all enzymes.2

Despite the availability of the structures of OMPDC in other organisms
(e.g. E. coli - 1EIX,
M. thermoautotrophicum - 1DV7,
B. subtilis - 1DBT,
S. cerevisiae - 1DQW)
in apo form and in complexes with UMP and its analogs,
including 6-hydroxyuridine 5'-monophosphate (BMP) and 6-azauridine 5'-monophosphate,3
the mechanism of decarboxylation of OMP to UMP remains uncertain.

We have solved the native structure of orotidine 5-monophosphate decarboxylase from
Plasmodium yoelii
PY01515,
Plasmodium berghei

NCBI accession XP_679107
and Plasmodium vivax

TIGR accession Pv111555.
All orthologs feature the signature DxKxxDIxxT motif in their catalytic sites
(DMKINDIGNT in both cases).

As shown below, the is strong sequence homology amongst the Plasmodium family of OMPDC,
even though each is only about 20% identical to the same enzyme in other organisms.
The P. berghei and P. yoelii enzymes are 85% identical,
with both being 68% identical to the Pf OMPDC
and about 50% identical to the P. vivax orthologue.
The orthologues from the human parasites, namely Pf and Pv,
are 62% identical.
As with other proteins, OMPDC from P. knowlesi is 82% identical to
the P. vivax enzyme and 62% identical to the P. falciparum orthologue.

PBG-PF10_0225 -HFKTKLKNRRNEVNTCLCIGLDPDEDDIKNFMRNEEKNGYKNVKNNMNSNNNRI-----------ENVIKIGKEILLTDEENIENLSEE PY01515 MHFKTKLKNRRSEVNTCLCIGLDPDEDDIKNFMKNEEQNGYKNIKNNMNSNNNGI-----------ENIIKIGKEILLTDGENIQNLSEE PF10_0225 MGFKVKLEKRRNAINTCLCIGLDPDEKDIENFMKNEKENNYNNIKKNLKEKY--------------INNVSIKKDILLKAPDNIIREEKS PKN-PF10_0225 MNLKTKLQKRRDEVNTCLCIGLDPDEADIKSFMQSEKQNGYQSIKKNLSNHGSSS--------QGGLFTPQVGGTMLLAENPPKE-VQEK PV-PF10_0225 -NLKIKLQKRRDEVNTCLCIGLDPDEADIKSFMQSEKQNGYQSVKKNLSNSGSSSSSSNSSSGKGELFAPQMGGQMLLAETPPKE-AQEK PBG-PF10_0225 DKFFYFFNHFCFYIINNTKEYALIYKMNFAFYIPYGSVGINALKNVFDYLNSMNIPTMLDMKINDIGNTVKNYRKFIFEYLKSDSCTINV PY01515 DKFFYFFNHFCFYIINNTKEYALVYKMNFAFYIPYGSVGINALKNVFDYLNSMNIPTMLDMKINDIGNTVKNYRKFIFEYLKSDSCTINV PF10_0225 EEFFYFFNHFCFYIINETNKYALTFKMNFAFYIPYGSVGIDVLKNVFDYLYELNIPTILDMKINDIGNTVKNYRKFIFEYLKSDSCTVNI PKN-PF10_0225 DEFFYFFNHFCFYIINETKQYALSYKMNFAFYLPYGSLGVDVLKNVFDYLHYLDVPTILDIKMNDIGNTVKHYS

Materials and Methods

Pb-OMPDC: Plasmodium orotidine 5

PDB:2FDS

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:68074385
Entry Clone Source:Plasmodium berghei genomic DNA
SGC Clone Accession:PBG-PF10_0225:; plate M:E7
Tag:N-terminal: His6-tag with integrated thrombin protease site: mgsshhhhhhssglvpr*gs
Host:E. coli BL21-(DE3)-CodonPlus-RIL from Stratagene

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsHFKTKLKNRRNEVNTCLCIGLDPDEDDIKNFMRNEEKNGYKNVKNNMNSNNNRIENVIKIGKEILLTDEENIENLSEEDKFFYFFNHFCFYIINNTKEYALIYKMNFAFYIPYGSVGINALKNVFDYLNSMNIPTMLDMKINDIGNTVKNYRKFIFEYLKSDSCTINVYMGTSMLKDICFDYEKNKYYSAYVLIKTTNKDSFIFQNELSINDKQAYIVMAEETQKMATDLKIDQNNEFIGFVVGSNAFEEMKIIRNKFPDSYILSPGIGAQNGDLYKTLKNGYNKDYEKLLINVGRAITKSPNPKKSSESYYNQIIQIFKDIENGGNIEQVYL
Vector:p28a-LIC

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 ~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 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. The volume of the Ni-NTA resin was pre-determined by the predicted protein yield from test expression analysis. 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 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 1Â5 mM after approximately 15 more minutes. The protein was dialyzed overnight against Crystal Buffer and finally concentrated using a 15 mL Amicon Ultra centrifugal filter device (Millipore). The concentrated sample was flash frozen in N2(l) and stored at -80 ºC.

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:74 mg/mL
Ligand
MassSpec:
Crystallization:The protein was crystallized by means of hanging drop vapor diffusion in a 24-well Linbro plate. The plate was set with 1.5 microL protein with NaI added to 20 mM and 1.5 microL buffer in each drop, and 300 microL reservoir volume per well. Crystals grew overnight in 16% PEG 3350 and 140 mM diammonium hydrogen citrate at 18 degC.
NMR Spectroscopy:
Data Collection:
Data Processing: