Human Hydroxyacid Oxidase 1

Target ID:

HAO1A

Aliases:

GOXGOX1HAOX1MGC142225MGC142227

Deposition Date:

Friday 24th November 2006

Families:

Oxidoreductases

Related Diseases:

Metabolism

Structure type:

apo

Download Coordinates:

2NZL

Authors:

E.UGOCHUKWU,K.KAVANAGH,E.PILKA,G.BERRIDGE,J.E.DEBRECZENI,E.PAPAGRIGORIOU,A.TURNBULL,F.NIESEN,O.GILEADI,F.VON DELFT,M.SUNDSTROM,C.H.ARROWSMITH,J.WEIGELT,A.EDWARDS,U.OPPERMANN,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2NZL

tabs

Structure Details

The metabolism of fatty acids proceeds through alpha- and beta-oxidation pathways, depending on the substrate. Whereas beta-oxidation occurs on straight alkyl-chains mostly in mitochondria, the metabolism of 3-methyl branched chain fatty acids proceeds through an initial alpha oxidation step, found in peroxisomes, followed by several rounds of beta oxidation. Although not all details of the alpha oxidation pathway are established yet, oxidation of 2-hydroxyacid intermediates generated in the alpha oxidation pathway to hydrogenperoxide and keto acids that are decarboxylated, is postulated. The recent discovery of 3 human 2-hydroxyacid oxidases (HAOX1-3) and their functional characterization as 2-hydroxyacid oxidases suggest an involvement of these peroxisomal enzymes in alpha oxidation pathways.

The HAOX enzymes belong to the FMN-dependent alpha-hydroxy acid dehydrogenase family, and are related to plant glycolate oxidases. The expression of HAOX1 in liver and kidney as well as the ability to oxidize glyoxylate to oxalate suggest an involvement of this enzyme in primary hyperoxaluria type 1.

Materials and Methods

HAO1 - Human Hydroxyacid Oxidase 1

PDB:2NZL

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NM_017545
Entry Clone Source:Origene
SGC Clone Accession:
Tag:N-terminal hexahistidine tag, mhhhhhhssgvdlgtenlyfqs
Host:BL21(DE3)-R3/Rosetta

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqsMLPRLIC INDYEQHAKSVLPKSIYDYYRSGANDEET LADNIAAFSRWKLYPRMLRNVAETDLSTS VLGQRVSMPICVGATAMQRMAHVDGELAT VRACQSLGTGMMLSSWATSSIEEVAEAGP EALRWLQLYIYKDREVTKKLVRQAEKMGY KAIFVTVDTPYLGNRLDDVRNRFKLPPQL RMKNFETSTLSFSPEENFGDDSGLAAYVA KAIDPSISWEDIKWLRRLTSLPIVAKGIL RGDDAREAVKHGLNGILVSNHGARQLDGV PATIDVLPEIVEAVEGKVEVFLDGGVRKG TDVLKALALGAKAVFVGRPIVWGLAFQGE KGVQDVLEILKEEFRLAMALSGCQNVKVI DKTLVRKNPLAVSKI
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:An overnight culture (20 ml) was used to inoculate 1L TB with 50 µg/ml of Kanamycin. The cells were cultured at 37°C until the OD reached 4.0 and then the temperature was decreased to 25°C. IPTG was added to 0.5 mM final concentration and the culture kept at 25°C overnight. Cells were harvested by centrifugation and the pellet stored at -80°C.

Purification

Procedure

Extraction

Procedure
The cell pellet from 1 L of culture was resuspended in 40ml of extraction buffer. The sample was homogenized by passing once through the Constant cell systems fluidizer, the DNA precipitated by adding 0.15% PEI and incubated on ice for 45mins and then centrifuged at 37000 xg. The supernatant was filtered through 0.2 m M serum acrodiscs and kept for further purification.
Concentration:
Ligand
MassSpec:The mass was verified by Mass spec to be correct within 1Da
Crystallization:Crystals were grown by vapor diffusion at 20°C. A sitting drop consisting of 50 nl of protein (12 mg/ml) and 100 nl well solution was equilibrated against well solution containing 30% PEG 1000, 0.1 M Malate-MES-Tris pH 7.0. The crystal was transferred to a cryo-protectant comprised of well solution supplemented with 20% ethylene glycol before flash-cooling in liquid nitrogen.
NMR Spectroscopy:
Data Collection:Resolution: 1.35 Å, X-ray source: Synchrotron SLS-X10SA, single wavelength.
Data Processing: