Human 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1

Target ID:

HMGCS1A

Aliases:

HMGCSMGC90332

Deposition Date:

Friday 23rd March 2007

Families:

Oxidoreductases

Related Diseases:

Metabolism

Structure type:

apo

Download Coordinates:

2P8U

Authors:

A.P.TURNBULL,N.SHAFQAT,E.SALAH,F.H.NIESEN,N.BURGESS,G.BUNKOCZI,J.DEBRECZENI,A.C.W.PIKE,C.UMEANO,F.GORREC,F.VONDELFT,J.WEIGELT,C.H.ARROWSMITH,M.SUNDSTROM,A.EDWARDS,U.OPPERMANN,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2P8U

tabs

Structure Details

The cytosolic enzyme 3-hydroxy 3 methylglutaryl coenzyme A synthase 1 catalyses the condensation of acetyl coenzyme A (Ac-CoA) with acetoacetyl Coenzyme A to yield 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA). HMG CoA is further reduced by HMG CoA reductase. This rate determining step is targeted by a cholesterol lowering class of drugs, the statins. This essential mammalian pathway is important for cholesterol and prenoid synthesis.

HMGCS1 gene expression is upregulated by the the antipsychotic drugs clozapine and haloperidol in cultured human glioma cells. Since several studies have reported on structural abnormalities, decreased myelination and oligodendrocyte dysfunction in post-mortem brains from schizophrenic patients HMGCS1 is one of the interesting etiological candidate targets in schizophrenia. The enzyme is widely expressed with highest expression found in liver and adrenal cortex. Furthermore, due to its location in the mevalonate pathway it may play a role in hypercholesterolanemia and atherosclerosis.

Materials and Methods

HMGCS1: Human 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1

PDB:2P8U

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:IMAGE:2819708
Entry Clone Source:MGC
SGC Clone Accession:HMGCS1A-c007
Tag:N-terminal His-tag with TEV protease cleavage site (Tag sequence in lower case):
Host:E. coli strain: BL21(DE3)-R3 pRARE2

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqsMDVGIVA LEIYFPSQYVDQAELEKYDGVDAGKYTIG LGQAKMGFCTDREDINSLCMTVVQNLMER NNLSYDCIGRLEVGTETIIDKSKSVKTNL MQLFEESGNTDIEGIDTTNACYGGTAAVF NAVNWIESSSWDGRYALVVAGDIAVYATG NARPTGGVGAVALLIGPNAPLIFERGLRG THMQHAYDFYKPDMLSEYPIVDGKLSIQC YLSALDRCYSVYCKKIHAQWQKEGNDKDF TLNDFGFMIFHSPYCKLVQKSLARMLLND FLNDQNRDKNSIYSGLEAFGDVKLEDTYF DRDVEKAFMKASSELFSQKTKASLLVSNQ NGNMYTSSVYGSLASVLAQYSPQQLAGKR IGVFSYGSGLAATLYSLKVTQDATPGSAL DKITASLCDLKSRLDSRTGVAPDVFAENM KLREDTHHLVNYIPQGSIDSLFEGTWYLV RVDEKHRRTYARRP
Vector:pNIC28-Bsa4

Growth

Medium:TB
Antibiotics:
Procedure:10 microL of a glycerol stock was inoculated into 5ml of TB medium (supplemented with 50 microG/ml Kanamycin, 34 microG/ml Chloramphenicol) and cultured at 37degC o/n in a shaking incubator (275 rpm). Next day 0.75 ml of o/n culture was used to inoculate 1 litre of TB medium (6x) and grown at 37degC with vigorous shaking (160 rpm) until the culture reaches an OD600 of 1.4. 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 -20degC until further use.

Purification

Procedure

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 .
Concentration:
Ligand
Acetyl-CoAMassSpec:Corresponds to theoretical mass, as determined by ESI-TOF MS.
Crystallization:Crystals were grown by vapor diffusion in sitting drops at 4degC. Before setting up the experiment acetyl-CoA was added to the protein to a final concentration of 5 mM. A sitting drop consisting of 50 nl protein and 100 nanoliter well solution was equilibrated against well solution containing 25% PEG 3350, 0.2 M ammonium sulfate, 0.1 M BIS-TRIS pH 6.5. The crystal was mounted in a loop directly from the drop and flash-cooled in liquid nitrogen.
NMR Spectroscopy:
Data Collection:
Data Processing: