Dehydrogenase domain of Human D-bifunctional Protein (D-BP)

Target ID:

HSD17B4A

Aliases:

MFE-2

Deposition Date:

Friday 8th April 2005

Families:

Oxidoreductases

Related Diseases:

SignallingNeurobiologyMetabolism

Structure type:

apo

Download Coordinates:

1ZBQ

Authors:

P.LUKACIK,N.SHAFQAT,K.KAVANAGH,J.BRAY,F.VON DELFT,A.EDWARDS,C.ARROWSMITH,M.SUNDSTROM,U.OPPERMANN,STRUCTURAL GENOMICSCONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

1ZBQ

tabs

Structure Details

The peroxisomal D-bifunctional protein (D-BP) has a tripartite structure
with different functional domains involved in peroxisomal beta-oxidation pathways
of 2-methyl branched fatty acids and very long chain fatty acids,
degradation of the bile acid intermediates di- and trihydroxycoprostanic acid
and the inactivation of estradiol and androgen metabolites.

Whereas the N-terminal oxidoreductase domain (HSD17B4) oxidizes
in an NAD+ dependent reaction steroids and fatty acids,
the central domain (amino acids 324â€“596) catalyzes specifically
dehydration of D-3-hydroxyacyl-coenzyme A derivatives.
The physiological function of the C-terminal sterol carrier protein domain (SCP2) is unclear at present.

Several clinical mutations of the HSD17B4 gene have been described
and lead to D-specific multifunctional protein deficiency.
This manifests as severe peroxisomal disorder of a Zellweger-like syndrome,
and is characterised by a high plasma concentration of long chain fatty acids and bile acids.
These features impair renal functions and neuronal development
(neuronal migration defects and degeneration, Stiff man syndrome).

Materials and Methods

17β-HSD4

PDB:1ZBQ

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi:4504505
Entry Clone Source:synthetic
SGC Clone Accession:
Tag:N-terminal: His-tag with integrated TEV protease site: mgsshhhhhhssgrenlyfqghm. C-terminal: gs
Host:E.coli BL21 (DE3)

Construct

Prelude:
Sequence:mgsshhhhhhssgrenlyfqghMGSPLRFDGRVVLVTGAGAGLGRAYALAFAERGALVVVNDLGGDFKGVGKGSLAADKVVEEIRRRGGKAVANYDSVEEGEKVVKTALDAFGRIDVVVNNAGILRDRSFARISDEDWDIIHRVHLRGSFQVTRAAWEHMKKQKYGRIIMTSSASGIYGNFGQANYSAAKLGLLGLANSLAIEGRKSNIHCNTIAPNAGSRMTQTVMPEDLVEALKPEYVAPLVLWLCHESCEENGGLFEVGAGWIGKLRWERTLGAIVRQKNHPMTPEAVKANWKKICDFENASKPQSIQESTGSIIEVLSKIDSgs
Vector:pET-11 derivative

Growth

Medium:
Antibiotics:
Procedure:Medium: LB with 100 mg/L ampicillin. 1 L LB in 2.5 L baffled flasks was inoculated with an overnight culture. The culture was grown at 37°C to OD=0.6-0.7. 1 mM IPTG was then added and incubation continued over a period of 4-5hours at 37°C. The cells were then collected by centrifugation and frozen at -20°C.

Purification

Procedure

Extraction

Procedure
Extraction buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 5 mM Imidazole, 1 mM PMSF, 0.5mM TCEP. Extraction buffer was added to frozen cells and the cells were resuspended. The cells were disrupted by sonication.
Concentration:
Ligand
MassSpec:
Crystallization:Hampton I screen condition 17: 0.2M lithium sulphate monohydrate, 0.1M TRIS hydrochloride pH 8.5, 30% w/v polyethylene glycol 4000. Vapour Diffusion, sitting Drop at temperature 293K
NMR Spectroscopy:
Data Collection:
Data Processing: