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Structure Details

GAMT: Human Guanidinoacetate N-methyltransferase

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PDB Code 1ZX0 Target Class Transferases

Target GAMT
Alias PIG2, TP53I2
Disease Area/Function metabolism
Date Deposited 2005-06-07
Authors A.DONG, H.WU, H.ZENG, P.LOPPNAU, M.SUNDSTROM, C.H.ARROWSMITH, A.M.EDWARDS, A.BOCHKAREV, A.N.PLOTNIKOV, STRUCTURAL GENOMICSCONSORTIUM (SGC)

Struc Details 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 the S-adenosylmethionine-dependent methyl transferases.

Guanidinoacetate N-methyltransferase catalyzes the last step of creatine biosynthesis. Creatine plays an important role in energy metabolism of brain and muscle. GAMT deficiency is associated with neurologic syndromes and muscular hypotonia. Biochemically it is characterized by low excretion of creatine, deficiency of creatine and creatine phosphate, and simultaneous accumulation of guanidinoacetate in brain. We have solved human guanidinoacetate N-methyltransferase structure in complex with S-adenosylhomocysteine at 1.9 Å resolution. The structure provides us important information for understanding of catalytic mechanism of creatine biosynthesis. It may also facilitate the evaluation of this protein as a potential therapeutic target.

Materials & Methods
StructureGAMT
PDB Code1ZX0
Entry clone accession GI:4503909
Entry clone source MGC
Tag N-terminal: His-tag with integrated thrombin protease site: MGSSHHHHHHSSGLVPRGS
Construct sequenceGSAPSATPIFAPGENCSPAWGAAPAAYDAADTHLRILGKPVMERWETPYMHALAAAASSKGGRVLEVGFGMAIAASKVQE
APIDEHWIIECNDGVFQRLRDWAPRQTHKVIPLKGLWEDVAPTLPDGHFDGILYDTYPLSEETWHTHQFNFIKNHAFRLL
KPGGVLTYCNLTSWGELMKSKYSDITIMFEETQVPALLEAGFRRENIRTEVMALVPPADCRYYAFPQMITPLVTKG
Vector p28a-LIC
Expression host E.coli BL21 (DE3) codon plus RIL (Stratagen)
Growth method GAMT was expressed in E.coli BL21 (DE3) codon plus RIL in Terrific Broth (TB) in the presence of 50 µg/mL of kanamycin at 37oC to an OD600 of 1.5. Cells were then induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM, and incubated overnight at 15oC.
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 (1 XPBS, 0.25 M NaCl, 5 mM imidazol, 2 mM ?-mercaptoethanol, 5% glycerol) with protease inhibitor (0.1mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
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 Tris-HCl buffer, pH 8.0, containing 250 mM NaCl and 50 mM imidazole, and the protein was eluted with elution buffer (20 mM Tris-HCl, pH 8.0, 250 mM NaCl, 250 mM imidazole). The protein was loaded on Superdex200 column (26x60) (Amersham Biosciences), equilibrated with 20 mM Tris-HCl buffer, pH 8.0, and 150 mM NaCl, at flow rate 4 mL/min. Thrombin (Sigma) was added to combined fractions containing GAMT and incubated overnight at 4oC. The protein was further purified to homogeneity by ion-exchange chromatography on Source 30Q column (10x10) (Amersham Biosciences), equilibrated with buffer 20 mM Tris-HCl, pH 8.0, and eluted with linear gradient of NaCl up to 500 mM concentration (20CV). Purification yield was 70 mg of the protein per 1L of culture.
Crystallization Purified GAMT was complexed with S-adenosyl-L-homocysteine (SAH) (Sigma) at 1:5 molar ratio of protein:SAH and crystallized using the hanging drop vapor diffusion method at 20 °C by mixing 1.5 µl of the protein solution with 1.5 µl of the reservoir solution containing 21% PEG3350, 0.1 M CaCl2,0.1 M BisTris, pH 6.5, 1 mM Guanidine HCl.