glutathione peroxidase 7

Target ID:

GPX7A

Aliases:

CL683FLJ14777GPX6NPGPx

Deposition Date:

Thursday 8th March 2007

Families:

Oxidoreductases

Related Diseases:

CancerMetabolism

Structure type:

apo

Download Coordinates:

2P31

Authors:

K.L.KAVANAGH,C.JOHANSSON,E.PAPAGRIGORIOU,G.KOCHAN,C.UMEANO,O.GILEADI,F.VON DELFT,J.WEIGELT,C.H.ARROWSMITH,M.SUNDSTROM,A.EDWARDS,U.OPPERMANN,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2P31

tabs

Structure Details

Glutathione peroxidase 7 (GPX7, NPGPX) is a recently identified non-selenocysteine containing phospholipid hydroperoxidase. At least seven glutathione peroxidases (GPX1?7) exist in mammalian cells: cytoplasmic GPX1, gastrointestinal GPX2, secreted GPX3, phospholipid hydroperoxidase GPX4, epididymal GPX5, olfactory GPX6, and non-selenocysteine containing phospholipid hydroperoxidase GPX7. The wild-type enzymes typically contain an active site selenocysteine that is encoded by the UGA stop codon. Replacement of this residue by a cysteine, as occurs in GPX7 and GPX5, dramatically decreases the catalytic activity. Although the majority of mammalian glutathione peroxidases are tetrameric, the phospholipid hydroperoxidases GPX7 and GPX4 are monomeric.

The GPX family constitutes an important mechanism in the protection against oxidative stress by scavenging and inactivating hydrogen and lipid peroxides to water or lipid hydroxyls in a glutathione-dependent reductive reaction. To date GPX7 has not been extensively characterized but it is reported to be cytoplasmic and widely expressed. A role for GPX7 in alleviating oxidative stress in breast cancers induced by dietary fatty acids is postulated (1).

Materials and Methods

GPX7: Human glutathione peroxidase 7

PDB:2P31

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC032788
Entry Clone Source:MGC
SGC Clone Accession:
Tag:TEV-cleavable (*), N-terminal histag.
Host:Rosetta-R3

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfq*SMQQEQD FYDFKAVNIRGKLVSLEKYRGSVSLVVNV ASECGFTDQHYRALQQLQRDLGPHHFNVL AFPCNQFGQQEPDSNKEIESFARRTYSVS FPMFSKIAVTGTGAHPAFKYLAQTSGKEP TWNFWKYLVAPDGKVVGAWDPTVSVEEVR PQITALVR
Vector:pNIC28-Bsa4

Growth

Medium:TB + 50 microG/ml Kanamycin + 34 µg/ml chloramp
Antibiotics:
Procedure:3 litre TB in 2.5-L baffled flasks were inoculated with 10 ml overnight culture and grown at 37degC. The protein expression was induced with 1 mM IPTG at OD600 = 4.6 at 18degC overnight. The cells were collected by centrifugation and frozen at -80degC.

Purification

Procedure
Ni-affinity:The cell extract was loaded on the column at 0.8 ml/minute on an AKTA-express system (GE/Amersham). The column was then washed with 10 volumes of lysis buffer, 10 volumes of wash buffer, and then eluted with elution buffer at 0.8 ml/min. The eluted peak of A280 was automatically collected.

Gel filtration: The eluted fractions from the Ni-affinity Histrap columns were loaded on the gel filtration column in GF buffer at 0.80 ml/min. Eluted proteins were collected in 2 ml fractions and analyzed on SDS-PAGE gels.

Concentration: DTT was added to the protein to a final concentration of 5 mM. The protein was concentrated in Amicon (5 K) to 12 mg/ml and stored at -80degC. The protein concentration was determined spectrophotometrically using the predicted molar extinction coefficient (26930 M-1cm-1).

Extraction

Procedure
Frozen cell pellets were thawed at 37°C and resuspended in a total volume of 150 ml lysis buffer. The cells were disrupted by high pressure (20 kpsi) and nucleic acids and cell debris removed by adding 0.15% PEI , followed by centrifugation for 30 minutes at 40,000xg. The supernatant was further clarified by filtration (0.45 µm).
Concentration:
Ligand
MassSpec:
Crystallization:Crystals were grown by vapor diffusion at 20degC. A sitting drop consisting of 75 nanoliter protein (12 mg/mL) and 75 nanoliter well solution was equilibrated against well solution containing 25% PEG 3350, 0.2 M ammonium sulfate, 17% glycerol. The crystal was mounted directly from the drop and flash-cooled in liquid nitrogen.
NMR Spectroscopy:
Data Collection:Resolution: 2.0Å; X-ray source: Synchrotron SLS-X10SA
Data Processing:

References

Utomo A, Jiang X, Furuta S, Yun J, Levin DS, Wang YC, Desai KV, Green JE, Chen PL, Lee WH. Identification of a novel putative non-selenocysteine containing phospholipid hydroperoxide glutathione peroxidase (NPGPx) essential for alleviating oxidative stress generated from polyunsaturated fatty acids in breast cancer cells.
J Biol Chem. 2004 Oct 15;279(42):43522.