Human Uridine monophosphate synthetase - Orotidine-5

Target ID:

UMPSA

Aliases:

OPRTUMPS

Deposition Date:

Friday 16th February 2007

Families:

Nucleotide metabolism

Related Diseases:

Cancer

Structure type:

apo

Download Coordinates:

2JGY

Authors:

M.MOCHE,D.OGG,C.ARROWSMITH,H.BERGLUND,R.BUSAM,R.COLLINS,L.G.DAHLGREN,A.EDWARDS,U.B.ERICSSON,S.FLODIN,A.FLORES,S.GRASLUND,M.HAMMARSTROM,B.M.HALLBERG,L.HOLMBERG-SCHIAVONE,I.JOHANSSON,T.KARLBERG,U.KOSINSKA,T.KOTENYOVA,L.LEHTIO,M.E.NILSSON,T.NYMAN,C.PERSSON,J.SAGEMARK,P.STENMARK,M.SUNDSTROM,J.UPPENBERG,M.UPSTEN,A.G.THORSELL,S.VAN DEN BERG,J.WEIGELT,P.NORDLUND,STRUCTURAL GENOMICSCONSORTIUM (SGC)

Site:

Stockholm

ICM Datapack:

ICM Datapack

2JGY

tabs

Structure Details

Uridine monophosphate synthetase (UMPS) performs the final two steps of UMP biosynthesis in mammals by an N-terminal phosphoribosyl transferase domain (EC:2.4.2.10) and a C-terminal Orotidine 5â€™-phosphate decarboxylase (ODCase) domain (EC:4.1.1.23). Orotic aciduria is a rare human disease associated with UMPS with phenotypic features such as megaloblastic anemia and crystalline orotic acid in the urine that can be corrected with uridine based dietary supplements [1]. ODCase is well studied with respect to reaction mechanism and many inhibitors are available from efforts in making cancer drugs during the 1960s and 1970s [2]. Recently 6-iodo-UMP was shown to irreversibly inhibit malarial ODCase creating hopes for a new class of malarial inhibitors [3]. The human structure might guide efforts in finding inhibitors selective for ODCase of the causative agent.

We have determined the 3D structure of the human ODCase domain in its apo form to 1.95 Ã… resolution. Human ODCase crystallizes in P212121 with a dimer in the asymmetric unit where each subunit has the TIM-barrel fold (figure 1). The structure was determined by molecular replacement using the S. cerevisiae ODCase (1DQW) as our search model.

We recently determined a product UMP complex to 2.0 Ã… resolution (PDB code 2V30). When UMP binds in the active site a large loop, which was disordered in the apo structure, closes the active site from the environment. Residues from both subunits of the dimer contribute in making hydrogen bonds with UMP and residues in the UMP binding pocket are quite conserved across species.

Materials and Methods

UMPS

PDB:2JGY

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC000364
Entry Clone Source:Mammalian Gene Collection
SGC Clone Accession:
Tag:N-terminal hexahistidine tag with integrated TEV protease cleavage site: mhhhhhhssgvdlgtenlyfq*s(m)
Host:BL21(DE3)

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqsmELSFGARAELPRIHPVASKLLRLMQKKETNLCLSADVSLARELLQLADALGPSICMLKTHVDILNDFTLDVMKELITLAKCHEFLIFEDRKFADIGNTVKKQYEGGIFKIASWADLVNAHVVPGSGVVKGLQEVGLPLHRGCLLIAEMSSTGSLATGDYTRAAVRMAEEHSEFVVGFISGSRVSMKPEFLHLTPGVQLEAGGDNLGQQYNSPQEVIGKRGSDIIIVGRGIISAADRLEAAEMYRKAAWEAYLSRLG
Vector:PNIC-BSA4

Growth

Medium:TB
Antibiotics:
Procedure:MB4933:
750 ml TB media supplemented with 50 µg/mL kanamycin was inoculated with 20 ml of the overnight cultures. The large scale cultivations were grown in tunair flasks 37°C until OD600 reaches approximately 1.8-2. The flasks were then moved to 18°C and induced with 0,5 mM IPTG after 1 hour. Cultures were allowed to grow overnight at 18°C.

Purification

Procedure
Columns:Columns:
Ni column: HiTrap IMAC HP
GF column:HiLoadÂ 16/60 Superdex 200 Prep Grade

Purification was performed on an ÄKTAprime. Prior to purification, columns were equilibrated with IMAC Bind/Wash1 Buffer (HiTrap Chelating) and Gel filtration buffer (Superdex 200). The protein sample was loaded on the HiTrap Chelating column that was washed with IMAC Bind/Wash1 Buffer followed by IMAC Wash2 Buffer. Bound protein was eluted from the IMAC columns with IMAC Elution Buffer and loaded in the Gel filtration column.

Extraction

Procedure
Harvest by centrifugation, rotor F8S-4X 1000y (=SLC-6000) at 5000 rpm for 10 minutes in 4°C and freeze the pellet at -80°C. Prior to purification the cell pellet was resuspended in Lysis Buffer. Cells were disrupted by sonication (4s on 4 off 3 min 80% amplitude) and samples were centrifuged for 20 min at 20500 rpm. The soluble fraction was filtered through 0.2 µm and subjected to further purification on the ÄKTAprime.
Concentration:23 mg/mL
Ligand
MassSpec:
Crystallization:The 23 g/L stock solution was diluted 1:1 to 11.5 g/L using the gel filtration buffer and mixed 0.1+0.1 µL with well solution consisting of 30% (w/v) PEG 3350 and 0.1 M HEPES pH 7.5 using a Phoenix crystallization robot from Art Robbins Instruments at 20ºC and then left in 4ºC for equilibration. A single crystal appeared somewhere in between day 3 and day 12 and was frozen in cryo solution consisting of 20% PEG3350, 20% glycerol and 0.1M Hepes pH 8.4.The product containing crystal was obtained in a drop of 0.2 µL protein+0.1 µL well solution consisting of 16% (w/v) PEG 3350 and 0.1 M HEPES pH 7.6. The crystal was soaked 15 minutes in a solution of 22% PEG3350, 0.1M HEPES pH 7.5, 20% Glycerol, 10mM uridine 5Â-monophosphate (UMP) before being flash cooled in liquid nitrogen.
NMR Spectroscopy:
Data Collection:For the apo-crystal X-ray data in space group P212121 (59.8, 77.8, 152.6, α = β = γ = 90) were collected at MaxLab (i911-2) and processed using XDS and XSCALE. The crystal soaked in UMP had the same space group and very similar cell dimensions; these data were collected at ESRF (id29) and processed using Mosflm and Scala of the CCP4 package to a resolution of 2 Å.
Data Processing:The apo structure was solved by molecular replacement using MOLREP with the Escherichia coli structure (1DQW) as our search model. The initial solution was then auto(re)built in ARP/WARP, which was possible due to the high quality of the 1.95 Å data. For both the apo and the UMP complex structure the final cycles of model building were performed in COOT with REFMAC5 as the refinement program.

References

Huguley, C. M., Jr., Bain, J. A., Rivers, S. L. & Scoggins, R. B. (1959). Refractory megaloblastic anemia associated with excretion of orotic acid. Blood 14, 615-34.
Miller, M. (2004). Insight into the catalytic mechanism of orotidine 5Â´-phosphate decarboxylase from crystallography and mutagenesis. In Topics in Current Chemistry, Vol. 238, pp. 43-62. Springer-verlag, Berlin Heidelberg.
Bello, A. M., Poduch, E., Fujihashi, M., Amani, M., Li, Y., Crandall, I., Hui, R., Lee, P. I., Kain, K. C., Pai, E. F. & Kotra, L. P. (2007). A potent, covalent inhibitor of orotidine 5'-monophosphate decarboxylase with antimalarial activity. J Med Chem 50, 915-21.