Human methionine adenosyltransferase I

Target ID:

MAT1A

Aliases:

MATMATA1SAMSSAMS1

Deposition Date:

Wednesday 20th December 2006

Families:

Oxidoreductases

Related Diseases:

MetabolismCancer

Structure type:

apo

Download Coordinates:

2OBV

Authors:

E.S.PILKA,N.SHAFQAT,K.L.KAVANAGH,C.COOPER,V.HOZJAN,A.TURNBULL,F.VON DELFT,C.H.ARROWSMITH,A.EDWARDS,J.WEIGELT,M.SUNDSTROM,U.OPPERMANN,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2OBV

tabs

Structure Details

S-adenosyl methionine, the principle methyl donor for transmethylation reactions, is synthesized by methionine adenosyl transferases (MATs) from methionine and ATP. In humans, there are two major methionine adenosyl transferases: MAT I/III which is liver specific and MAT II which is ubiquitously expressed. MAT I and MATIII are homo-oligomers composed of 4 and 2 catalytic α1 subunits, respectively. The MAT II isozyme is a hetero-oligomer consisting of two catalytic α2 subunits and a regulatory β subunit. The catalytic subunits are essential enzymes that are highly conserved evolutionarily; the β subunit is present only in mammals and by associating α2 with alters its kinetic properties.

Mutations in MAT1 lead to methionine-adenosyltransferase deficiency, which is characterized as an inborn error of metabolism resulting in isolated hypermethioninemia. Most patients have no clinical abnormalities, although some neurological abnormalities have been reported in rare cases with severe loss of enzyme activity; and brain demyelination has been observed in some cases. Patients with alcoholic liver disease and cirrhosis show a H2O2-induced MAT inactivation and abnormal methionine metabolism. Furthermore, increased MAT1A activity has been found in colorectal carcinoma and levels of MAT1A expression is correlated with the stages of the colorectal tumor.

MAT1A is antiapoptotic in normal hepatocytes, but proapoptotic in liver cancer cells making it an attractive agent for both chemoprevention and treatment of hepatocellular carcinoma.

Materials and Methods

MAT1 - Human methionine adenosyltransferase I

PDB:2OBV

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:MAT1A-s001 (gi|4557737)
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal His-tag with TEV protease cleavage site (Tag sequence in lowercase)
Host:Rosetta-R3

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqsMGVFMFT SESVGEGHPDKICDQISDAVLDAHLKQDP NAKVACETVCKTGMVLLCGEITSMAMVDY QRVVRDTIKHIGYDDSAKGFDFKTCNVLV ALEQQSPDIAQCVHLDRNEEDVGAGDQGL MFGYATDETEECMPLTIILAHKLNARMAD LRRSGLLPWLRPDSKTQVTVQYMQDNGAV IPVRIHTIVISVQHNEDITLEEMRRALKE QVIRAVVPAKYLDEDTVYHLQPSGRFVIG GPQGDAGVTGRKIIVDTYGGWGAHGGGAF SGKDYTKVDRSAAYAARWVAKSLVKAGLC RRVLVQVSYAIGVAEPLSISIFTYGTSQK TERELLDVVHKNFDLRPGVIVRDLDLKKP IYQKTACYGHFGRSEFPWEVPRKLVF
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:10 microL of a glycerol stock was inoculated into 100ml of TB medium (supplemented with 50µg/ml kanamycin ) and cultured at 37°C o/n in a shaking incubator (275 rpm). Next day 12 ml of o/n culture was used to inoculate 1 litre of TB medium (4x) and grown at 37°C with vigorous shaking (160 rpm) until the culture reaches an OD600 of 1.0. Temperature was reduced to 18°C, and cells were induced with IPTG at a concentration of 0.5 mM, and further cultivated for 16 hrs. Cells were harvested by centrifugation at 6500 rpm for 10 min, and the cell pellet was stored at -20°C until further use.

Purification

Procedure
Column 1 : Ni-Sepharose 6 Fast Flow
Procedure: The column was packed with 2 ml of Ni-Sepharose 6 Fast Flow slurry and equilibrated with 15 ml of binding buffer. The supernatant was loaded onto the column and the column was washed with 20 ml of binding buffer and then 20 ml of washing buffer. The protein was eluted with 10 ml of elution buffer.

Column 2 : SuperDex 200 16/60 HiLoad (GE/Amersham)
Procedure: The eluted protein from the Ni-affinity column was loaded on the gel filtration column in GF buffer at 1.0 ml/min on an AKTA Purifier system. Eluted proteins were collected in 2 ml fractions.

Column 3: HP Q column (ion exchange).
Procedure: The MAT1A-k003 was applied to 5ml HP Q column in buffer A and eluted from the column by a linear gradient with buffer B.

Concentration : 16.04 mg/ml using Vivaspin 10K concentrators

Extraction

Procedure
Complete® protease inhibitors (Roche, 1 tbl/50 ml). 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 .
Concentration:
Ligand
MassSpec:Corresponds to theoretical mass, as determined by ESI-TOF MS .
Crystallization:Crystals were grown by vapor diffusion at 20°C. Prior to crystallization 5 mM S-adenosyl methionine was added to the protein. A sitting drop consisting of 100 nl protein and 50 nl well solution was equilibrated against well solution containing 200 mM NaF, 20 % PEG 3350, 10 % ethylene glycol.
NMR Spectroscopy:
Data Collection:Resolution: 1.9Å; X-ray source: Rotating anode, Rigaku FR-E superbright .
Data Processing: