Human selenocysteine lyase

Target ID:

SCLYA

Aliases:

SCLSCLY

Deposition Date:

Wednesday 21st June 2006

Families:

AA metabolic enzymes

Related Diseases:

Metabolism

Structure type:

apo

Download Coordinates:

Superceded by 3GZC

Authors:

R.COLLINS,M.HOGBOM,C.ARROWSMITH,H.BERGLUND,A.EDWARDS,M.EHN,S.FLODIN,A.FLORES,S.GRASLUND,B.M.HALLBERG,M.HAMMARSTROM,T.KARLBERG,T.KOTENYOVA,P.NILSSON-EHLE,P.NORDLUND,T.NYMAN,D.OGG,C.PERSSON,J.SAGEMARK,P.STENMARK,M.SUNDSTROM,A.G.THORSELL,J.UPPENBERG,S.VAN DEN BERG,J.WEIGELT,L.HOLMBERG-SCHIAVONE,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Stockholm

ICM Datapack:

ICM Datapack

2HDY

tabs

Structure Details

Selenium is an essential trace element that is incorporated into proteins in a unique way in the form of selenocysteine, the 21st amino acid. At least 25 human proteins (not counting splice variants) have been discovered that contain selenocysteine. Several of them are involved in redox metabolism and anti-oxidative cellular pathways.

Selenocysteine lyases (EC 4.4.1.16) are NifS-like pyridoxal-5′-phosphate-dependent enzymes that catalyze the interconversion between L-selenocysteine and hydrogen selenide + L-alanine. The enzyme is thus located at the branch point between alanine and selenoaminoacid metabolism and is also suggested to be involved in delivery of selenium to selenophosphate synthetase. Selenophosphate is the selenium containing precursor molecule in the synthesis of selenocystyl-tRNA for use in selenoprotein biosynthesis. The human enzyme is, unlike several bacterial homologues, specific for selelenocysteine and does not act on the sulphur analogue cysteine.

We have solved the structure of the human selenocysteine lyase to 2.1 Ã… resolution (PDB entry 2HDY). The protein is a homodimer with the PLP cofactor covalently bound to Lys 259. The active site is situated close to the dimer interface and both monomers contribute to the active site cavity. Interestingly, one part of the structure displays large differences between the two monomers in the asymmetric unit. This loop contains conserved residues important for activity in other selenocysteine lyases and cysteine desulfurases. In one of the monomers this loop restricts active site access while in the other it is extended and opens access to the active site.

Materials and Methods

SCLY

PDB:2HDY

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:AAH07891
Entry Clone Source:
SGC Clone Accession:
Tag:
Host:

Construct

Prelude:
Sequence:SMGRDAPAPAASQPSGCGKHNSPERKVYMDYNATTPLEPEVIQAMTKAMWEAWGNPSSPYSAGRKAKDIINAARESLAKMIGGKPQDIIFTSGGTESNNLVIHSVVKHFHANQTSKGHTGGHHSPVKGAKPHFITSSVEHDSIRLPLEHLVEEQVAAVTFVPVSKVSGQTEVDDILAAVRPTTRLVTIMLANNETGIVMPVPEISQRIKALNQERVAAGLPPILVHTDAAQALGKQRVDVEDLGVDFLTIVGHKFYGPRIGALYIRGLGEFTPLYPMLFGGGQERNFRPGTENTPMIAGLGKAAELVTQNCEAYEAHMRDVRDYLEERLEAEFGQKRIHLNSQFPGTQRLPNTCNFSIRGPRLQGHVVLAQCRVLMASVGAACHSDHGDQPSPVLLSYGVPFDVARNALRLSVGRSTTRAEVDLVVQDLKQAVAQLEDQA
Vector:pNIC-Bsa4

Growth

Medium:
Antibiotics:
Procedure:30 microL competent BL-21 (DE3) cells were transformed with 2 µl plasmid miniprep for 30 min on ice followed by heatshock at 42°C for 45 sec. SOC, 125 µl, was added to the cellsuspension which was then incubated for 1 hour at 37°C and plated on LB-plates containing kanamycin (50 µg/mL). 20 mL TB with 100 µg kanamycin/mL was inoculated with cells and grown overnight (ON) at 30°C. The inoculation culture was added to 1.5 L TB (supplemented with 50 µg kanamycin/mL) in 2 L bottles. The flask was incubated in the LEX system-water bath at 37°C until OD600 reached 1. At this time the flask was transferred to an 18°C water bath in the LEX-system. Expression of protein was induced by addition of 0.5 mM IPTG and continued for approximately 18 hours.
Cells were harvested by centrifugation in a SLC-6000 rotor for 10 minutes at 5000 rpm (OD600 9.1; WCW 23.3 g). Pellets were suspended in 100 ml 50 mM Nafosfate pH 7.5, 10 % glycerole, 0.5 mM TCEP and 500 mM NaCl, 10mM imidazole and Complete EDTA-free protease inhibitor (Roche Biosciences). Suspended cells were stored at -80°C until further use. Before lysis, 4 µl of 250U/µl benzonase (Novagen) was added to the suspended cells.

Purification

Procedure
SCLYA was purified on a Hi-Trap chelating column followed by a Superdex 200 gel filtration column on the Äkta Express. The protein migrated as a well defined single peak and fractions containing protein were coloured bright yellow from the bound PLP co-factor. These fractions were pooled and the TCEP-concentration adjusted to 2 mM before concentration in AmiconUltra filters to 22.8 mg/mL. The protein was aliquoted and quick-frozen in liquid nitrogen.

TEV-cleavage: TEV-cleavage was performed by adding 422 µl His-tagged TEV (30 µM) to 30 mg protein in a total volume of 1.8 ml in gel filtration buffer with 2 mM TCEP for approximately 72 hours at 4°C. The sample was diluted with 20 mM Hepes, 500 mM NaCl, 10% glycerole and 0.5 mM TCEP at pH 7.5 before loading it onto a His-trap crude column on an Äkta Prime. The cleaved protein bound weakly to the column and was eluted with the same buffer plus 35 mM imidazole. After elution the TCEP concentration was adjusted to 2 mM and the sample concentrated to 35.6 mg/ml in a AmiconUltra MWCO 30000 concentrator.

Extraction

Procedure
The sample was sonicated (Sonics VibraCell) at 80% amplitude for 3 min (effective time, pulse: 4Â on 4Â off).The sample was spun for 30 min at 20500 rpm in a Sorvall SA-800 rotor and the soluble fraction decanted and filtered through a 0.45 µm syringe filter
Concentration:
Ligand
MassSpec:
Crystallization:Sitting drops containing 0.1 µl of protein solution (17 mg/mL) + 0.1µl well solution containing 100 mM HEPES pH 6.7 and 10% PEG6000 was left to equilibrate against well solution. Crystals appeared after 3 days at 20 degC.
NMR Spectroscopy:
Data Collection:Data was collected at the ESRF, beam-line ID23-1. Data was processed with XDS and XSCALE. The space group is P212121 with cell parameters 59 86 189 90 90 90.
Data Processing:The structure was solved by Molecular Replacement using a monomer of the E. coli cysteine desulfurase IscS (pdb entry: 1P3W) as the search model. The asymmetric unit contains one protein dimer. Refmac was used for refinement and Coot for model building. TLS refinement with 4 TLS groups was used in Refmac. The final model starts at glutamate 29 and ends at glutamine 444, the penultimate residue in the sequence. Residues 120 to 132 in both monomers are disordered and invisible in the electron density.

References

Esaki N, Nakamura T, Tanaka H, Soda K. (1982) Selenocysteine lyase, a novel enzyme that specifically acts on selenocysteine. Mammalian distribution and purification and properties of pig liver enzyme. J. Biol. Chem. 257; 4386-91.

Daher R, Van Lente F. (1992)Characterization of selenocysteine lyase in human tissues and its relationship to tissue selenium concentrations. J Trace Elem Electrolytes Health Dis. Sep;6(3):189-94.

Mihara H, Kurihara T, Yoshimura T, Esaki N. (2000) Kinetic and mutational studies of three NifS homologs from Escherichia coli: mechanistic difference between L-cysteine desulfurase and L-selenocysteine lyase reactions. J Biochem (Tokyo). 2000 Apr;127(4):559-67.

Tamura T, Yamamoto S, Takahata M, Sakaguchi H, Tanaka H, Stadtman TC, Inagaki K. (2004) Selenophosphate synthetase genes from lung adenocarcinoma cells: Sps1 for recycling L-selenocysteine and Sps2 for selenite assimilation. Proc. Natl. Aacd. Sci. USA, Nov 16;101(46):16162-7.