Human glucosamine-6-phosphate N-acetyltransferase 1 in complex with CoA and N-acetyl-D-glucosamine-6-phosphate

Target ID:

GNPNAT1

Aliases:

FLJ10607GNPNATGpnat1

Deposition Date:

Thursday 27th July 2006

Families:

Transferases

Related Diseases:

Metabolism

Structure type:

apo

Download Coordinates:

2O28

Authors:

J.MIN,H.WU,H.ZENG,P.LOPPNAU,J.WEIGELT,M.SUNDSTROM,C.H.ARROWSMITH,A.M.EDWARDS,A.BOCHKAREV,A.N.PLOTNIKOV,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

2O28

tabs

Structure Details

The Gcn5-related N-acetyltransferases (GNATs) represent a large superfamily of functionally diverse enzymes that catalyze the transfer of an acetyl group from AcetylCoA to the primary amine of a wide range of substrates. Glucosamine-6-phosphate N-acetyltransferase 1 (GNPNAT1, GNA1) is an amino-sugar N-acetyltransferase member of the GNAT superfamily.

GNPNAT1 is the key player in the pathway toward de novo synthesis of the essential metabolite UDP-GlcNAc. The enzyme is present in various eukaryotic organisms. In mouse, the homologue of GNPNAT1, EMeg32, is essential for embryonic development and inactivation of this gene in mouse embryonic fibroblasts leads to resistance to apoptotic stimuli and defects in cell proliferation. In Saccharomyces cerevisiae, GNA1 has been shown to control multiple cell cycle steps and to be essential for the survival ofyeast. In addition, GNA1 mutants in Candida albicans exhibit reduced virulence when injected into mice, making GNA1 a potential target for the development of antifungal agents.

Human GNPNAT1 gene is located on chromosome 14 (14q22.1). The 184 amino acids protein is associated with Golgi and endosomal membranes. We have solved the crystal structure of human GNPNAT1 in complex with CoA and N-Acetyl-D-glucosamine-6-phosphate at 1.8 Å resolution. The structure provides important information not only on substrate binding, but also on catalytic mechanism.

Materials and Methods

GNPNAT1-CoA-GlcNAc6P

PDB:2O28

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:37620194
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal: His-tag with integrated TEV protease site: MHHHHHHSSGRENLYFQ*G
Host:BL21 (DE3) codon plus RIL (Stratagene)

Construct

Prelude:
Sequence:gMKPDETPMFDPSLLKEVDWSQNTATFSPAISPTHPGEGLVLRPLCTADLNRGFFKVLGQLTETGVVSPEQFMKSFEHMKKSGDYYVTVVEDVTLGQIVATATLIIEHKFIHSCAKRGRVEDVVVSDECRGKQLGKLLLSTLTLLSKKLNCYKITLECLPQNVGFYKKFGYTVSEENYMCRRFLK
Vector:p28-MHL

Growth

Medium:
Antibiotics:
Procedure:GNPNAT1 was expressed in E.coli BL21 (DE3) codon plus RIL in Terrific Broth (TB) in the presence of 50 µg/ml of kanamycin. Cell were grown at 37oC to an OD600 of 1.5 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM, and incubated overnight at 15oC.

Purification

Procedure
The crude extract was cleared by centrifugation. The clarified lysate was loaded onto 5 ml HiTrap Chelating column (Amersham Biosciences), charged with Ni²⁺. The column was washed with 10 CV of 20 mM HEPES, pH 7.4, containing 500 mM NaCl and 50 mM imidazole, 5% glycerol, and the protein was eluted with elution buffer (20 mM HEPES, pH 7.4, 500 mM NaCl, 250 mM imidazole, 5% glycerol). The protein was dialyzed against 20 mM HEPES, pH 7.4, 500 mM NaCl, 5% glycerol in the presence of TEV protease. The dialyzed protein was passed through a 5 ml Ni HiTrap column and loaded on Superdex200 column (26x60) (Amersham Biosciences), equilibrated with 20 mM PIPES buffer, pH 6.5, and 250 mM NaCl, at flow rate 4 ml/min. The pooled fractions containing GNPNAT1 was further purified to homogeneity by ion-exchange chromatography on Source 30S column (10x10) (Amersham Biosciences), equilibrated with buffer 20 mM PIPES pH 6.5, and eluted with linear gradient of NaCl up to 0.5 M concentration (20CV). Purification yield was 25 mg of the protein per 1L of culture.

Extraction

Procedure
Cells were harvested by centrifugation at 7,000 rpm. The cell pellets were frozen in liquid nitrogen and stored at -80°C. For the purification the cell paste was thawed and resuspended in lysis buffer (50 mM HEPES, pH 7.4, 0.5 M NaCl, 5 mM imidazol, 2 mM ß-mercaptoethanol, 5% glycerol) with protease inhibitor (0.1 mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:59 mg/ml
Ligand
MassSpec:Expected mass=20806.1 Da, measured mass =20831.1 Da
Crystallization:Purified GNPNAT1 was complexed with Coenzyme A and N-Acetyl-D-glucosamine-6-phosphate(Sigma) at 1:5 molar ratio of protein: CoA/ N-Acetyl-D-glucosamine-6-phosphate and crystallized using the hanging drop vapor diffusion method by mixing 2 microL of protein solution with 2 microL of the reservoir solution containing 27% PEG3350, 0.2 M MgCl2, 0.1 M Bicine, pH 9.0.
NMR Spectroscopy:
Data Collection:
Data Processing: