Human glutathione peroxidase 3

Target ID:

GPX3A

Aliases:

GPx-PGPX3GSHPx-3GSHPx-P

Deposition Date:

Wednesday 29th August 2007

Families:

Oxidoreductases

Related Diseases:

MetabolismCancerChromatin and epigenetics

Structure type:

apo

Download Coordinates:

2R37

Authors:

Pilka, E.S., Guo, K., Gileadi, O., Rojkowa, A., von Delft, F., Pike, A.C.W., Kavanagh, K.L., Johannson, C., Sundstrom, M., Arrowsmith, C.A., Weigelt, J., Edwards, A., Oppermann, U.

Site:

Oxford

ICM Datapack:

ICM Datapack

2R37

tabs

Structure Details

The reactions carried out by the glutathione peroxidase (GPX) family constitute 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. At least seven glutathione peroxidases (GPX1-7) exist in mammalian cells: cytoplasmic GPX1, gastrointestinal GPX2, plasma 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 which normally signals translation termination. The 3' UTR of Sec-containing genes have a common stem-loop structure, the sec insertion sequence (SECIS), which is necessary for the recognition of UGA as a Sec codon rather than as a stop signal. Replacement of this residue by a cysteine, as occurs in GPX7 and GPX5, dramatically decreases the catalytic activity.

GPX3, like the majority of mammalian glutathione peroxidases, is tetrameric. This cytosolic enzyme is expressed in liver, kidney, heart and gonads, and is also found in plasma, serum, and cerebrospinal fluid. Deregulated GPX3 levels/activities are found in a variety of diseases: increased levels were found in asthma, HIV-infected patients, heart of diabetic mouse. Low levels were found in plasma of children with chronic renal failure and patients with prostate cancer. GPX3 activity is also severely reduced in seminal fluid of infertile males and may be a new useful marker of gonadal function in men.

The crystal structure confirms a tetrameric form of GPX3. The active site SelCys73 was mutated to Gly to facilitate protein production in E. coli.

Materials and Methods

GPX3

PDB:2R37

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|6006001
Entry Clone Source:MGC collection, active-site mutant: Se-Cys (U) was replaced by G
SGC Clone Accession:GPX3A-c068
Tag:C-terminal TEV-cleavable (at *) his-tag with the following sequence AENLYFQ*SHHHHHHDYKDDDDK
Host:BL21(DE3)-R3-pRARE2

Construct

Prelude:
Sequence:MQEKSKMDCHGGISGTIYEYGALTIDGEEYIPFKQYAGKYVLFVNVASYGGLTGQYIELNALQEELAPFGLVILGFPCNQFGKQEPGENSEILPTLKYVRPGGGFVPNFQLFEKGDVNGEKEQKFYTFLKNSCPPTSELLGTSDRLFWEPMKVHDIRWNFEKFLVGPDGIPIMRWHHRTTVSNVKMDILSYMRRQAALGVAENLYFQ*SHHHHHHDYKDDDDK
Vector:pNIC-CTHF

Growth

Medium:TB
Antibiotics:
Procedure:10ml of overnight culture was added into 1L TB with 50 µg/ml of Kanamycin & 34 µg/ml of Chloramphenicol (total 3 L). The cells were cultured at 37°C until the OD reached 1.511 and then decreased the temperature to 18°C. IPTG was added at 0.5mM (final concentration) and kept the culture at 18°C for overnight.

Purification

Procedure
Column 1 : Ni-Sepharose
Column 2 : Ni-Sepharose

The column was packed by 4 ml of Ni-NTA slurry and equilibrated with 15 ml of binding buffer. The supernatant was loaded onto the column and the flow through was collected. The column was washed with 50 ml of washing buffer I and then 5 ml of washing buffer II. The protein was eluted with 5 ml of elution buffer I and II respectively and 8ml of elution buffer III.

Enzymatic treatment: 100 m l of TEV protease (6mg/ml) were added into the sample. The sample was incubated at 4°C overnight. His-tag was cleaved by TEV protease. The sample was loaded onto the column (packed from 0.4 ml 0f Ni-Sepharose slurry). The flow through was collected and the column was then washed with 3 mls of the buffers with 5, 10 & 20 mM Imidazole (also collected).

Extraction

Procedure
The cells were harvested by centrifugation at 4,000 g for 10 min. The pellet from 1 L culture was resuspended in 25 ml of extraction buffer. The sample was homogenized by using the EmulsiFlex-05 homogenizer (Glen Creston) and then centrifuged at 37505 g. The supernatant was kept for further purification.
Concentration:36.34 mg/ml.
Ligand
MassSpec:23560.1 (23527 expected)
Crystallization:Crystals were grown by vapor diffusion at 4°C in 150nl sitting drops. The drops were prepared by mixing 100nl of protein solution and 50nl of precipitant consisting of 0.1M PCB pH 8.0, 60% MPD. Crystals were flash-cooled in liquid nitrogen.
NMR Spectroscopy:
Data Collection:1.85 Å; X-ray source: Synchrotron SLS-X10SA.
Data Processing: