Human phytanoyl-CoA dioxygenase domain containing 1

Target ID:

PHYD1B

Aliases:

MGC16638

Deposition Date:

Tuesday 30th January 2007

Families:

Oxidoreductases

Related Diseases:

Metabolism

Structure type:

apo

Download Coordinates:

2OPW

Authors:

Z.ZHANG,D.BUTLER,M.A.MCDONOUGH,K.L.KAVANAGH,J.E.BRAY,S.S.NG,F.VON DELFT,C.H.ARROWSMITH,J.WEIGELT,A.EDWARDS,M.SUNDSTROM,C.J.SCHOFIELD,U.OPPERMANN,STRUCTURAL GENOMICS CONSORTIUM(SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2OPW

tabs

Structure Details

PHYD1 is a member of the iron containing human 2-oxoglutarate (2â€™OG) dependent oxygenase family, members of which are involved in a variety of oxidative reactions acting on a spectrum of different substrates. The reactions carried out by many members of this family comprise hydroxylation reactions acting on molecules such as DNA or proteins. Protein modifications include oxidation of amino acids such as aspartyl or prolyl residues, or demethylation reactions of methylated lysyl residues of histones. A subset of the 2â€™OG family is involved in carnitine synthesis pathways or acts on small molecules such as fatty acyl CoAs.

The PHYD1 gene is located on human chromosome 9q34 and has been identified as member of the 2â€™OG family. Sequence comparison shows that it has highest homology to phytanoyl-CoA hydroxylase (PHYH), a peroxisomal enzyme critical to degradation of phytanoyl CoA, a metabolite of chlorophyll. Whereas several PHYH mutations associated to severe clinical phenotypes have been identified, PHYD1 has to this end not been investigated.

The structure of PHYD is similar to the structure of the recently determined PHYH structure, suggesting similar substrate specificities.

Materials and Methods

Vector: pET24a. Details [PDF]; Sequence [FASTA] or [GenBank]

Tag and addition: None

Protein sequence:
MACLSPSQLQKFQQDGFLVLEGFLSAEEC
VAMQQRIGEIVAEMDVPLHCRTEFSTQEE
EQLRAQGSTDYFLSSGDKIRFFFEKGVFD
EKGNFLVPPEKSINKIGHALHAHDPVFKS
ITHSFKVQTLARSLGLQMPVVVQSMYIFK
QPHFGGEVSPHQDASFLYTEPLGRVLGVW
IAVEDATLENGCLWFIPGSHTSGVSRRMV
RAPVGSAPGTSFLGSEPARDNSLFVPTPV
QRGALVLIHGEVVHKSKQNLSDRSRQAYT
FHLMEASGTTWSPENWLQPTAELPFPQLY
T

Host: E.coli BL21(DE3)

Cell Growth: Cells were grown at 370C in 2 x TY medium including 15 µg/ml kanamycin with shaking speed 200 rpm. Protein expression was induced by adding 0.5 mM IPTG at OD₅₅₀=1.4. Cells were grown for another 4 hours and then harvested by centrifugation.

Cell lysis: The cell pellet was resuspended in lysis buffer containing 50 mM HEPES, 10% glycerol, 2 mM DTT, 3 mM EDTA, pH 7.5. Lysozyme and DNase I were added to the cell suspension at 1 mg/ml and 20 µg/ml respectively, and the suspension was incubated at 40°C for 20 minutes. Cells were lysed by sonication followed by centrifugation to remove cell debris. The clarified supernatant was loaded onto the first column in the next step.

Column 1: DEAE Sepharose fast flow 50/25 150 ml bedvolume ion-exchange column. The column was equilibrated with buffer A containing 50 mM HEPES, 10% glycerol, 2mM DTT, 2 mM EDTA and pH7.5.

Procedure: The supernatant was loaded onto the column and washed with 3 column volume of buffer A. PHYD1 was eluted using a linear gradient from 0% to 30% buffer B (buffer A containing 1 M NaCl) in 6 column volumes. Fractions containing PHYD1 were pooled and concentrated.

Column 2: Superdex-75 700 ml gel-filtration column.

Procedure: The concentrated fractions containing the PHYD1 was loaded onto the gel filtration column and eluted with buffer C containing 25 mM Tris, 10% glycerol, 1mM DTT, 2 mM EDTA and pH 7.5. The fractions containing PHYD1 as judged by SDS-PAGE were pooled and subjected to a final step of purification.

Column 3: Mono Q 16/10 ion-exchange column.

Procedure: The pooled fractions were loaded directly to the Mono Q column pre-equilibrated with buffer D containing 25 mM Tris, 10% glycerol, 1 mM DTT, 1 mM EDTA and pH 8.0. PHYD1 was eluted with a gradient from 0 to 15% of buffer E (buffer D containing 1 M NaCl) in 5 column volumes. The fractions containing PHYD1 were pooled, concentrated, aliquoted and stored at -80°C for crystallographic and enzyme activity assay studies.

Mass spectrometry: The ion-electronspray mass spectrometry measurement of the sample gives molecular weight of 32280 Da comparing to the calculated one of 32411 Da, suggesting that the n-terminal methionine is removed after translation.

Crystallisation: Crystal screens were initially set up with sitting drop using 100 nl protein plus 100 nl well solution. Crystals were obtained with the screening condition of 0.2 M calcium acetate, 0.2 M potassium chloride 20% PEG 3350. Macro seeding is then used to obtain single and better crystals for structure studies. The Cryoprotectant used the mother solution with glycerol added to a final concentration 20%

Data collection, Resolution: 1.9 Å, space group hexagonal P3(1)21, X-ray source: Synchrotron SLS-X10SA, single wavelength.