Human nicotinamide N-methyltransferase

Target ID:

NNMT

Deposition Date:

Wednesday 27th September 2006

Families:

Transferases

Related Diseases:

Metabolism

Structure type:

apo

Download Coordinates:

2IIP

Authors:

G.BERNSTEIN,J.MIN,H.WU,W.TEMPEL,H.ZENG,P.LOPPNAU,G.V.AVVAKUMOV,G.WASNEY,J.WEIGELT,M.SUNDSTROM,C.H.ARROWSMITH,A.M.EDWARDS,A.BOCHKAREV,A.N.PLOTNIKOV,STRUCTURAL GENOMICSCONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2IIP

tabs

Structure Details

The methylation of a wide variety of biologically active molecules is a common theme in biology.
The functional roles of methylation are wide ranging and include biosynthesis, metabolism, detoxification, signal transduction, protein sorting and repair, nucleic acid processing, gene silencing and imprinting.
The majority of methylation reactions are carried out by S-adenosylmethionine-dependent methyl transferases.

Nicotinamide N-methyltransferase belongs to the NNMT/PNMT/TEMT family. It catalyzes the N-methylation of nicotinamide and other pyridines to form pyridinium ions. This activity is important for biotransformation of many drugs and xenobiotic compounds. In human, NNMT is predominantly expressed in the liver. A lower expression is seen in the kidney, lung, skeletal muscle, placenta and heart. No expression detected in the brain or pancreas. We have solved the crystal structure of human nicotinamide N-methyltransferase in complex with S-adenosylhomocysteine at 2.05 Å resolution. The structure provides important information for understanding of catalytic mechanism of N-methylation of nicotinamide. At the mean time, we also solved the crystal structure of mouse NNMT in complex with S-adenosylhomocysteine at 1.8 Å. The two proteins share 84% sequence identity and their structures are also very similar. The monomers of human and mouse NNMT can be superimposed with R.M.S.D. of 0.551.

Materials and Methods

NNMT

PDB:2IIP

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:66933018
Entry Clone Source:MGC
SGC Clone Accession:NNMT_01:K100A:E101A:E103A:D7-GBC003
Tag:His-tag with integrated thrombin protease site: MGSSHHHHHHSSGLVPRGS
Host:E.coli BL21 (DE3) codon plus RIL (Stratagene)

Construct

Prelude:
Sequence:MESGFTSKDTYLSHFNPRDYLEKYYKFGSRHSAESQILKHLLKNLFKIFCLDGVKGDLLIDIGSGPTIYQLLSACESFKEIVVTDYSDQNLQELEKWLKaaPaAFDWSPVVTYVCDLEGNRVKGPEKEEKLRQAVKQVLKCDVTQSQPLGAVPLPPADCVLSTLCLDAACPDLPTYCRALRNLGSLLKPGGFLVIMDALKSSYYMIGEQKFSSLPLGREAVEAAVKEAGYTIEWFEVISQSYSSTMANNEGLFSLVARKLSRPL
Vector:p28a-LIC

Growth

Medium:
Antibiotics:
Procedure:NNMT mutant K100A/E101A/E103A was expressed in E.coli BL21 (DE3) codon plus RIL in 6L Terrific Broth (TB) in the presence of 50 µg/ml of kanamycin at 37oC to an OD600 of 1.0. Cells were then 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 at ~75000 x g for 60 minutes. The clarified lysate was loaded onto 3 ml Ni-NTA column (Qiagen). The column was washed with 5 CV of 50 mM Tris-HCl buffer, pH 8.0, containing 500 mM NaCl, 5% glycerol and 25 mM imidazole, and the protein was eluted with elution buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 250 mM imidazole, 5 % glycerol). The protein was loaded on Superdex200 column (26x60) (Amersham Biosciences), equilibrated with 50 mM Tris-HCl buffer, pH 8.0, and 500 mM NaCl, 5% glycerol, 1 mM DTT, at flow rate 3.5 ml/min. Combined fractions containing NNMT mutant K100A/E101A/E103A were dialyzed against 4 L of 50 mM Tris, pH 8.0, 100 mM NaCl, 5% glycerol, 1 mM DTT overnight at 4oC. The protein was further purified to homogeneity by passing through HiTrap CaptoQ 5 ml column (Amersham Biosciences), equilibrated with buffer 50 mM Tris-HCl, pH 8.0, 5% glycerol, 1 mM DTT. The desired protein was in unbound protein fraction. Purification yield was 12.8 mg of the protein per 1L of culture.

Extraction

Procedure
Cells were harvested by centrifugation. The cell pellets were frozen in liquid nitrogen and stored at -80°C. For the purification the cell paste from 6L of cells was thawed and resuspended in lysis buffer (50 mM Tris, pH 8.0, 0.5 M NaCl, 5 mM imidazol, 2 mM ß-mercaptoethanol, 5% glycerol) with protease inhibitor (1mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:17.2 mg/ml
Ligand
MassSpec:expected MW is 31427.0 Da, measured MW is 31372.09 Da
Crystallization:Purified NNMT was complexed with 2.5 mM S-adenosyl-L-homocysteine (SAH) (Sigma) and crystallized using the sitting drop vapor diffusion method at 18 °C by mixing 0.5 µl of the protein solution with 0.5 µl of the reservoir solution containing 2.15 M ammonium sulfate, 0.1 M HEPES/Na, pH 7.2.
NMR Spectroscopy:
Data Collection:
Data Processing: