Human GDP-L-fucose synthase with bound NADP

Target ID:

TSTA3A

Aliases:

FXP35BTSTA3

Deposition Date:

Monday 29th April 2013

Families:

Oxidoreductases

Related Diseases:

CancerMetabolismSignalling

Structure type:

protein-ligand

Download Coordinates:

4BKP

Authors:

M.Vollmar,N.Shafqat,A.Rojkova,T.Krojer,A.Bradley,J.W.Raynor,K.Kavanagh,F.von Delft,C.H.Arrowsmith,C.Bountra,A.Edwards,U.Oppermann,W.W.Yue

Site:

Oxford

4BKP

tabs

Structure Details

L-fucose is a 6-deoxy monosaccharide widely distributed in glycoconjugates across the bacterial, plant and animal kingdoms. In humans L-fucose is an essential sugar component in glycoproteins including the ABO blood group antigens and the cell adhesion family of selectins, proteins which play an important role in cell-to-cell communication and immune response [1]. L-Fucose is incorporated into proteins by the action of fucosyltransferases, using the activated sugar nucleotide GDP-L-fucose as donor. The inability to fucosylate proteins causes the immune disorder leukocyte adhesion deficiency type II in humans [2]. GDP-L-fucose is biosynthesized in a de novo metabolic pathway involving three enzymatic steps. The first step is catalysed by GDP-mannose 4,6 dehydratase (GMD), which converts GDP-D-mannose to GDP-4-keto-6-deoxy- D-mannose. This enolate intermediate is then subjected to epimerisation and reduction reactions, both steps being carried out by a single polypeptide, GDP-L-fucose synthase (GFS) [3].

GFS carries out its first enzymatic function on the GDP-4-keto-6-deoxy-D-mannose intermediate, namely the epimerisation at positions C3 and C5 of the sugar moiety. As a result, the alcohol group at C3 as well as the methyl group at C5 change their orientation from upwards phasing β-orientation to a downward phasing α-orientation. The key reactivity during the epimerisation reaction is the keto functionality at C4, introduced in the preceding reaction by GMD. After the epimerisation, GFS reduces the carbonyl at C4 in an NADPH-dependent manner, and generates an alcohol group with a downward phasing β orientation. The resultant product, GDP-L-fucose, enters the Golgi apparatus and functions as a precursor for fucosylation.

GFS belongs to extended class of the short-chain dehydrogenase/reductase (SDR) enzyme superfamily, known to take part in the metabolism of diverse substrates and involve a myriad of chemical reactions [4]. Human GFS was first identified as the unannotated NADP(H)-binding protein FX enriched in erythrocytes, and later demonstrated to be the human homologue of a previously-characterized mouse protein known as tissue-specific transplantation antigen P35B (TSTA3). We have recently determined the structure of human GFS bound with NADP and GDP in two crystal forms (PDB codes 4B8W, 4B8Z). Here we report two follow-on structures of human GFS, namely a ternary product complex with NADP and GDP-fucose (4BL5), and as well as a holo complex with NADP (4BKP).

Materials and Methods

Molecular Biology

Entry Clone Accession: IMAGE:2819332
Entry Clone Source: MGC
SGC Construct ID: TSTA3A-c013
Protein Region: S7-K321
Vector: pNIC28-Bsa4
Tag: N-6HIS;N-TEV
Host: BL21(DE3)-R3

Sequence (with tag(s)): MHHHHHHSSGVDLGTENLYFQSMRILVTGGSGLVGKAIQKVVADGAGLPGEDWVFVSSKDADLTDTAQTRALFEKVQPTHVIHLAAMVGGLFRNIKYNLDFWRKNVHMNDNVLHSAFEVGARKVVSCLSTCIFPDKTTYPIDETMIHNGPPHNSNFGYSYAKRMIDVQNRAYFQQYGCTFTAVIPTNVFGPHDNFNIEDGHVLPGLIHKVHLAKSSGSALTVWGTGNPRRQFIYSLDLAQLFIWVLREYNEVEPIILSVGEEDEVSIKEAAEAVVEAMDFHGEVTFDTTKSDGQFKKTASNSKLRTYLPDFRFTPFKQAVKETCAWFTDNYEQARK
Sequence after tag cleavage: SMRILVTGGSGLVGKAIQKVVADGAGLPGEDWVFVSSKDADLTDTAQTRALFEKVQPTHVIHLAAMVGGLFRNIKYNLDFWRKNVHMNDNVLHSAFEVGARKVVSCLSTCIFPDKTTYPIDETMIHNGPPHNSNFGYSYAKRMIDVQNRAYFQQYGCTFTAVIPTNVFGPHDNFNIEDGHVLPGLIHKVHLAKSSGSALTVWGTGNPRRQFIYSLDLAQLFIWVLREYNEVEPIILSVGEEDEVSIKEAAEAVVEAMDFHGEVTFDTTKSDGQFKKTASNSKLRTYLPDFRFTPFKQAVKETCAWFTDNYEQARK
DNA Sequence: CATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGCGGATTCTAGTGACAGGGGGCTCTGGGCTGGTAGGCAAAGCCATCCAGAAGGTGGTAGCAGATGGAGCTGGACTTCCTGGAGAGGACTGGGTGTTTGTCTCCTCTAAAGACGCCGATCTCACGGATACAGCACAGACCCGCGCCCTGTTTGAGAAGGTCCAACCCACACACGTCATCCATCTTGCTGCAATGGTGGGGGGCCTGTTCCGGAATATCAAATACAATTTGGACTTCTGGAGGAAAAACGTGCACATGAACGACAACGTCCTGCACTCGGCCTTTGAGGTGGGCGCCCGCAAGGTGGTGTCCTGCCTGTCCACCTGTATCTTCCCTGACAAGACGACCTACCCGATAGATGAGACCATGATCCACAATGGGCCTCCCCACAACAGCAATTTTGGGTACTCGTATGCCAAGAGGATGATCGACGTGCAGAACAGGGCCTACTTCCAGCAGTACGGCTGCACCTTCACCGCTGTCATCCCCACCAACGTCTTCGGGCCCCACGACAACTTCAACATCGAGGATGGCCACGTGCTGCCTGGCCTCATCCACAAGGTGCACCTGGCCAAGAGCAGCGGCTCGGCCCTGACGGTGTGGGGTACAGGGAATCCGCGGAGGCAGTTCATATACTCGCTGGACCTGGCCCAGCTCTTTATCTGGGTCCTGCGGGAGTACAATGAAGTGGAGCCCATCATCCTCTCCGTGGGCGAGGAAGATGAGGTCTCCATCAAGGAGGCAGCCGAGGCGGTGGTGGAGGCCATGGACTTCCATGGGGAAGTCACCTTTGATACAACCAAGTCGGATGGGCAGTTTAAGAAGACAGCCAGTAACAGCAAGCTGAGGACCTACCTGCCCGACTTCCGGTTCACACCCTTCAAGCAGGCGGTGAAGGAGACCTGTGCTTGGTTCACTGACAACTACGAGCAGGCCCGGAAGTGACAGTAAAGGTGGATACGGATCCGAA

Protein Expression

Medium: LB
Antibiotics: Kanamycin
Procedure: The plasmid was transformed into E. coli, strain BL21 (DE3)-R3-pRARE. For protein expression 5 µl of a glycerol stock were used to inoculate 50 ml LB medium containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol. A starter culture was grown over night at 37°C and used on the following day to inoculate LB medium containing 50 µg/ml kanamycin. Cells were grown at a temperature of 37°C to a OD₆₀₀ of approx. 1.0 whereupon temperature was reduced to 18°C and cells were induced with 0.2 mM IPTG. Expresion continued overnight at 18^oC. Cells were harvested by centrifugation at 4°C at 6500 rpm for 11min. The supernatant was discarded and cell pellets stored at -20°C until further purification.

Protein Purification

Procedure: Cells were slowly thawed on ice, resuspended in lysis buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5 % (w/v) glycerol, 20 mM imidazole, 0.5 mM TCEP, 1 mM PMSF) and homogenised using an Avestin C-5 fluidizer. Insoluble material was removed by centrifugation (4°C, 16500 rpm, 45min. The supernatant was purified by Nickel affinity (Ni-sepharose; wash buffer: 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 5% glycerol, 30 mM imidazole; elution buffer: 50 mM HEPES pH 7.5, 50 mM NaCl, 5% glycerol, 250 mM imidazole) and size exclusion (Superdex S200; gel filtration buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 1mM PMSF, 0.5 mM TCEP) chromatography. Protein was concentrated to 12 to 15 mg/ml and stored at -80^oC.
Mass-spec Verification: Yes

Structure Determination

Crystallization: Crystals were grown by vapour diffusion in sitting drop at 20°C by setting up 12.6 mg/ml of protein in the presence of 5 mM NADP+. Rod shaped crystals appeared in a sitting drop consisting of 100 nl protein and 50 nl well solution which had been equilibrated against 20 µl well solution containing 20% PEG 3350, 0.1 M citrate pH 5.5. Crystals were mounted in the presence of 25% ethylene glycol and flash cooled in liquid nitrogen.
Data Collection: Beamline: Dmnd I03; Resolution: 2.7 Å
Data Processing: Data set was processed with XDS and merged and scaled with Aimless. Phases were derived by running Phaser and using chain B of 4B8Z as search model and four molecules were found in the asu creating two dimers. The model was further improved by subsequent model building in Coot and refinement with Refmac5.

References

Becker DJ, Lowe JB (2003) Fucose: Biosynthesis and biological function in mammals.
Yakubenia S and Wild MK (2006) Leukocyte adhesion deficiency II: Advances and open questions. FEBS J 273:4390-4398
Lau STB, Tanner ME (2008) Mechanism and Active Site Residues of GDP-Fucose Synthase. J Am. Chem Soc 130:17593-17602
Persson, B., et al (2009) The SDR (short-chain dehydrogenase/reductase and related enzymes) nomenclature initiative. Chem Biol Interact, 178:94-98