Human Sepiapterin Reductase

Target ID:

SPRA

Aliases:

INCLUDED

Deposition Date:

Wednesday 23rd March 2005

Families:

Oxidoreductases

Related Diseases:

NeurobiologyMetabolismDrug metabolism and toxicology

Structure type:

apo

Download Coordinates:

1Z6Z

Authors:

E.UGOCHUKWU,K.KAVANAGH,S.NG,C.ARROWSMITH,A.EDWARDS,M.SUNDSTROM,F.VON DELFT,U.OPPERMANN,STRUCTURAL GENOMICSCONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

1Z6Z

tabs

Structure Details

Sepiapterin reductase (SPR) is a member of the short-chain dehydrogenase/reductase family
and catalyzes the NADPH-dependent reduction of 6-pyruvoyl-tetrahydropterin to tetrahydrobiopterin (BH4).
This reaction constitutes an essential step to supply the cofactor for enzymes
such as nitric oxide synthase, phenylalanine-3-hydroxylase,
and the rate-limiting catecholamine and serotonin-biosynthesis enzymes tyrosine-4-hydroxylase
and tryptophan-5-hydroxylase.
Several mutations in the SPR gene (locus 2p14-2p12) were identified
in patients presenting with progressive psychomotor retardation,
dopa-sensitive dystonia, and severe dopamine and serotonine deficiencies.

Materials and Methods

SPR

PDB:1Z6Z

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi:4507185
Entry Clone Source:Synthetic
SGC Clone Accession:
Tag:mgsshhhhhhssgrenlyfq*gh (N terminus); gs (C terminus); TEV protease cleavable at *
Host:BL21 (DE3)

Construct

Prelude:
Sequence:EGGLGRAVCLLTGASRGFGRTLAPLLASLLSPGSVLVLSARNDEALRQLEAELGAERSGLRVVRVPADLGAEAGLQQLLGALRELPRPKGLQRLLLINNAGSLGDVSKGFVDLSDSTQVNNYWALNLTSMLCLTSSVLKAFPDSPGLNRTVVNISSLCALQPFKGWALYCAGKAARDMLFQVLALEEPNVRVLNYAPGPLDTDMQQLARETSVDPDMRKGLQELKAKGKLVDCKVSAQKLLSLLEKD
Vector:p11

Growth

Medium:
Antibiotics:
Procedure:Starter culture in 3 ml of LB + 100 µg/mL. Terrific Broth + 100 µg/mL, with in-house autoinduction componenets. Cultures grown for 7 hours at 37 ºC and the temperature reduced to 15 ºC overnight.

Purification

Procedure
Affinity column: 1 mL His-Trap (GE/Amersham). Buffers: 50 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 5 mM imidazole; 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 5% Glycerol, 30 mM imidazole; 50 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 250 mM imidazole. Procedure: AKTA Xpress Affinity/Gel Filtration

Gel filtration: SuperDex 200 16/60 HiLoad (GE/Amersham). Buffers: 10 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol. Procedure: AKTA Xpress Affinity/Gel Filtration

Extraction

Procedure
50 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 5 mM imidazole, PMSF to 1 mM added. Cell pellet was resuspended in the above buffer then frozen, the thawed resuspension was lysed using Avestin C-5 microfluidizer, 4 passes. PEI pH 8 added to a final concentration of 0.05% and spun in a JA-17 rotor at 17,000 rpm.
Concentration:
Ligand
MassSpec:
Crystallization:Affinity column: 1 mL His-Trap (GE/Amersham). Buffers: 50 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 5 mM imidazole; 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 5% Glycerol, 30 mM imidazole; 50 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 250 mM imidazole. Procedure: AKTA Xpress Affinity/Gel Filtration

Gel filtration: SuperDex 200 16/60 HiLoad (GE/Amersham). Buffers: 10 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol. Procedure: AKTA Xpress Affinity/Gel Filtration
NMR Spectroscopy:
Data Collection:
Data Processing: