human pyrroline-5-carboxylate synthetase 1 - aldehyde dehydrogenase domain

Target ID:

PYCSA

Aliases:

GSASMGC117316P5CSPYCS

Deposition Date:

Friday 26th May 2006

Families:

Oxidoreductases

Related Diseases:

MetabolismNeurobiologySignalling

Structure type:

apo

Download Coordinates:

2H5G

Authors:

E.PAPAGRIGORIOU,N.SHAFQAT,A.P.TURNBULL,G.BERRIDGE,V.HOZJAN,K.KAVANAGH,O.GILEADI,C.SMEE,J.BRAY,F.GORREC,M.SUNDSTROM,C.ARROWSMITH,J.WEIGELT,A.EDWARDS,U.OPPERMANN,STRUCTURALGENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2H5G

tabs

Structure Details

The synthesis of the amino acids proline, ornithine and arginine is initiated by a two step process consisting of phosphorylation and an NADPH-dependent reduction of the phosphorylated intermediate, by using glutamate or N-acetyl glutamate as initial substrates.

PYCS is involved in the initial steps of proline synthesis, and is a bifunctional enzyme with two distinct enzyme domains. The kinase domain, which is located at the N-terminus of PYCS, catalyzes the phosphorylation of glutamate to γ-glutamyl phosphate. This intermediate is then reduced to the glutamate-γ-semialdehyde product by the C-terminal oxidoreductase domain of PYCS. The resulting semialdehyde product of this reaction spontaneously cyclizes to form pyrroline-5-carboxylate, which is reduced to proline by a subsequent enzymatic step carried out by the distinct enzyme pyrroline carboxylate reductase.

Mutations in PYCS have been thus far identified only in the kinase domain, and lead to hyperammonemia and deficiencies in ornithine, citrullin, arginine and proline levels, and may be associated with neurodegeneration, cataracts and diseases of connective tissues.

The oxidoreductase domain of PYCS is an atypical member of the aldehyde dehydrogenase (ALDH) superfamily, and is classified as ALDH18A1.

Materials and Methods

PYCS: human pyrroline-5-carboxylate synthetase 1 - aldehyde dehydrogenase domain

PDB:2H5G

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:PYCSA-s001 ( gi|21361368)
Entry Clone Source:Origene
SGC Clone Accession:
Tag:N-terminal His-tag with TEV protease cleavage site (Tag sequence in lowercase)
Host:E.coli strain Rosetta

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqsMVKPAGP TVEQQGEMARSGGRMLATLEPEQRAEIIH HLADLLTDQRDEILLANKKDLEEAEGRLA APLLKRLSLSTSKLNSLAIGLRQIAASSQ DSVGRVLRRTRIAKNLELEQVTVPIGVLL VIFESRPDCLPQVAALAIASGNGLLLKGG KEAAHSNRILHLLTQEALSIHGVKEAVQL VNTREEVEDLCRLDKMIDLIIPRGSSQLV RDIQKAAKGIPVMGHSEGICHMYVDSEAS VDKVTRLVRDSKCEYPAACNALETLLIHR DLLRTPLFDQIIDMLRVEQVKIHAGPKFA SYLTFSPSEVKSLRTEYGDLELCIEVVDN VQDAIDHIHKYGSSHTDVIVTEDENTAEF FLQHVDSACVFWNASTRFSDGYRFGLGAE VGISTSRIHARGPVGLEGLLTTKWLLRGK DHVVSDFSEHGSLKYLHENLPIPQRNTN
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:10 microL of a glycerol stock was inoculated into 5ml of LB medium (supplemented with Kanamycin, 50µg/ml) in a 15 ml culture tube and cultured at 37°C o/n in a shaking incubator (275 rpm). The starter culture was used to inoculate 100 ml of LB medium and was grown at 37°C (200 rpm). At an OD of 2.2 the culture was harvested and the cell pellet was washed twice with M9 minimal medium (Molecular Dimensions Ltd). The cells were resuspended and used to inoculate 1 liter of prewarmed minimal medium. Methionine synthesis was suppressed by addition of leucine, isoleucine and valine (dissolved as 50 mg/l for each aa) and lysine, threonine, and phenylalanine (100mg/l of each aa). Selenomethionine was added to a concentration of 25 mg/l, and the cultures were induced by supplementation with 1 mM IPTG. Cells were grown overnight at 18°C, collected by centrifugation and stored frozen until further use.

Purification

Procedure
Column : 1 ml HisTrap crude (GE/Amersham)

Concentration: 20 mg/ml using Vivaspin 10K concentrators

Extraction

Procedure
Frozen cell pellets were thawed and resuspended in a total volume of 30-40 ml of lysis buffer, and disrupted by using Avestin C-5 microfluidizer, and a supernatant containing the target protein was obtained by centrifugation at 21,000 (rpm) for 45 minutes . The clear supernantant was passed twice over a 2.5 ml Ni-NTA resin, washed and eluted with the specified buffers.
Concentration:
Ligand
MassSpec:Corresponds to theoretical mass, as determined by ESI - TOF MS .
Crystallization:Crystals were grown by vapor diffusion at 20°C in 1.6M MgSO4 and 0.1M MES pH6.5. Ethylene Glycol (20% final concentration) was used as a cryoprotectant.
NMR Spectroscopy:
Data Collection:Resolution: 2.25Å X-ray source: Synchrotron SLS -X10
Data Processing: