orotidine-monophosphate-decarboxylase, putative

Target ID:

Pv-PF10_0225

Deposition Date:

Monday 1st May 2006

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

2GUU

Authors:

A.DONG,J.LEW,Y.ZHAO,E.SUNDARARAJAN,G.WASNEY,M.VEDADI,I.KOEIERADZKI,A.M.EDWARDS,C.H.ARROWSMITH,J.WEIGELT,M.SUNDSTROM,E.F.PAI,L.KOTRA,A.BOCHKAREV,R.HUI,M.AMANI,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

2GUU

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

Pv-OMPDC-6-aza-UMP

PDB:2GUU

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:Pv111555
Entry Clone Source:Plasmodium vivax Salvador I gDNA (kindly donated by John Barnwell of CDC)
SGC Clone Accession:PV-PF10_0225:; plate G:D12
Tag:N-terminal His-tag with integrated Thrombin protease site: mgsshhhhhhssglvprgs
Host:BL21 (DE3) CodonPlus-RIL from Strategene

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsNLKIKLQKRRDEVNTCLCIGLDPDEADIKSFMQSEKQNGYQSVKKNLSNSGSSSSSSNSSSGKGELFAPQMGGQMLLAETPPKEAQEKDEFFYFFNHFCFYIINETKEYALAYKMNFAFYLPYGSLGVDVLKNVFDYLHHLNVPTILDIKMNDIGNTVKHYRKFIFDYLRSDSCTANIYMGTQMLRDICLDEECKRYYSTFVLVKTTNADSHIFQNRLSLDGKEAYVVIAEEAQKMAKQLHLEENGEFVGFVVGANCYDEIKKIRELFPDCYILAPGVGAQKGDLRKMLCNGYSKNYEKVLINVGRAITKSGSPQQAAREYHQQIKEVLAELQE
Vector:pET28A-LIC

Growth

Medium:Terrific Broth (TB)
Antibiotics:50 microG/mL kanamycin and 25 microG/mL chloramphenicol
Procedure:Terrific Broth (TB)50 μg/mL kanamycin and 25 μg/mL chloramphenicolA 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 ~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 cell lysate was loaded onto a column containing 10 g DE-52 resin from Whatman (previously activated with 2.5 M NaCl and equilibrated with Binding Buffer) and then directly onto a 3 mL Ni-NTA (Qiagen) column. When all the lysate was loaded, the two column system was washed with 20 mL binding buffer. The Ni-NTA column was then washed with 200 mL of Wash Buffer. After washing, the protein was eluted from the Ni-NTA column with 15-20 mL of Elution Buffer. EDTA was added immediately to 1 mM; and DTT was added to 1 mM 15 minutes later.

The protein was put in a dialysis cassette (Pierce) for overnight dialysis in Crystal Buffer. The following day the protein was concentrated using a 15 mL Amicon Ultra centrifugal filter device (Millipore). Finally aliquots of the purified PY02252 protein were labeled 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 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.
Concentration:
Ligand
MassSpec:
Crystallization:The protein was crystallized by means of sitting drop vapor diffusion in a 96-well Intelli-Plate. The plate was set with 1.0 microL protein and 1.0 microL buffer in each drop, and 100 microL reservoir volume per well. Crystals grew overnight in 30% PEG 4K, 0.2M Sodium acetate, 0.1M Tris HCl, pH 8.5 at 18 ºC.
NMR Spectroscopy:
Data Collection:
Data Processing: