Human mitochondrial ketoacyl synthase

Target ID:

FLJ20604A

Aliases:

FASN2DFLJ20604KS

Deposition Date:

Tuesday 13th December 2005

Families:

Oxidoreductases

Related Diseases:

Metabolism

Structure type:

apo

Download Coordinates:

2C9H

Authors:

G.BUNKOCZI,X.WU,C.SMEE,O.GILEADI,C.ARROWSMITH,A.EDWARDS,M.SUNDSTROM,J.WEIGELT,F.VON DELFT,U.OPPERMANN

Site:

Oxford

ICM Datapack:

ICM Datapack

2C9H

tabs

Structure Details

The recently discovered mitochondrial fatty acid synthesis pathway consists of 5 enzymatic steps, including malonyl/acyl:ACP transfer, ketoacyl synthesis, ketoacyl reduction, dehydration, and enoyl-ACP reduction.
The mitochondrial pathway resembles in many aspects the bacterial counterpart in terms of organization as single enzymatic proteins.
This is clearly opposed to the cytosolic fatty acid synthase protein which is a single, dimeric multidomain protein of about 500 kDa.

Mitochondrial fatty acid synthesis is essential for endogenous production of lipoic acid, an important prosthetic group for mitochondrial enzymes and for maintenance of the mitochondrial redox status.

FLJ20604 is the recently identified human mitochondrial ketoacyl synthase and the structure was solved at a resolution of 2.0 Å.

Materials and Methods

FLJ20604A

PDB:2C9H

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:FLJ20604A-c010
Entry Clone Source:
SGC Clone Accession:
Tag:N-terminal Histag fusion to FLJ20604 with the following sequence: mhhhhhhssgvdlgtenlyfqsm
Host:BL21(DE3)- R3

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqsmRLHRRVVITGIGLVTPLGVGTHLVWDRLIGGESGIVSLVGEEYKSIPCSVAAYVPRGSDEGQFNEQNFVSKSDIKSMSSPTIMAIGAAELAMKDSGWHPQSEADQVATGVAIGMGMIPLEVVSETALNFQTKGYNKVSPFFVPKILVNMAAGQVSIRYKLKGPNHAVSTACTTGAHAVGDSFRFIAHGDADVMVAGGTDSCISPLSLAGFSRARALSTNSDPKLACRPFHPKRDGFVMGEGAAVLVLEEYEHAVQRRARIYAEVLGYGLSGDAGHITAPDPEGEGALRCMAAALKDAGVQPEEISYINAHATSTPLGDAAENKAIKHLFKDHAYALAVSSTKGATGHLLGAAGAVEAAFTTLACYYQKLPPTLNLDCSEPEFDLNYVPLKAQEWKTEKRFIGLTNSFGFGGTNATLCIAGL
Vector:pNIC28-BSA4

Growth

Medium:
Antibiotics:
Procedure:The cells were grown at 30°C in 1 liter Overnight Expression Instant TB Medium (Novagen) containing 50 µg/ml kanamycin for 26 hours before the cells were harvested by centrifugation.

Purification

Procedure
Column 1 : Ni_NTA batch column (Qiagen), 5 ml of 50% slurry in 1.5 x 10 cm column.
Buffers: Washing buffer: 500 mM NaCl, 5% glycerol, 50 mM Tris-HCl pH 7.5, 30 mM imidazole, 2 mM TCEP. Elution buffer: 500 mM NaCl, 5% glycerol, 50 mM HEPES pH 7.5, 250 mM imidazole, 2mM TCEP.
Procedure: After the supernatant was loaded to the Ni-NTA column equilibrated with the extraction buffer, the column was washed with 100ml washing buffer, and FLJ20604A was eluted in 15 ml elution buffer.

Column 2 : Hi Load 16/60 Superdex 200
Buffers : 300 mM NaCl, 10% glycerol, 10 mM HEPES pH 8.0, 2mM TCEP

Procedure: The eluted fraction was loaded to the Superdex column to change the buffer and achieve homogenous FLJ20604A for crystallization. The protein was concentrated using a 30000 MW cutoff Amicon Ultra concentration device.

Extraction

Procedure
Extraction buffer: 500 mM NaCl, 5% glycerol, 50 mM HEPES pH 7.5, 5 mM imidazole, 2 mM TCEP. Cell pellets from 1 liter were resuspened in 50 ml extraction buffer and then lysed by use of a French Press. The lysate was centrifuged at 18,000 RPM for 1 hour. The cleared lysate was loaded to the Ni-NTA column.
Concentration:
Ligand
MassSpec:
Crystallization:Column 1 : Ni_NTA batch column (Qiagen), 5 ml of 50% slurry in 1.5 x 10 cm column.
Buffers: Washing buffer: 500 mM NaCl, 5% glycerol, 50 mM Tris-HCl pH 7.5, 30 mM imidazole, 2 mM TCEP. Elution buffer: 500 mM NaCl, 5% glycerol, 50 mM HEPES pH 7.5, 250 mM imidazole, 2mM TCEP.
Procedure: After the supernatant was loaded to the Ni-NTA column equilibrated with the extraction buffer, the column was washed with 100ml washing buffer, and FLJ20604A was eluted in 15 ml elution buffer.

Column 2 : Hi Load 16/60 Superdex 200
Buffers : 300 mM NaCl, 10% glycerol, 10 mM HEPES pH 8.0, 2mM TCEP