EC Number   |
|---|
    1.4.1.2 | - |
| 1.4.1.2 | 3D structure determined by X-ray diffraction method, refined at a resolution of 2.9 A with a crystallographic R-factor of 19.9%. Crystals belonging to the space group P2(1)2(1)2(1) are grown in hanging drops in which 0.005 ml NAD+ is equilibrated against a reservoir containing 24% v/v polyethylene glycol monomethylether 550, 100 mM NaCl and 100 mM Tris-HCl |
| 1.4.1.2 | chimaeric protein consisting of domain I from NAD+-dependent GDH of Clostridium symbiosum, residues 1-200, domain II from NADP+-dependent GDH of Escherichia coli, residues 201-404 and the C-terminal helix again from Clostridium symbiosum, residues 405-448 which re-enters domain I. Domain II maintains its structural and functional integrity independently of the hinge and domain I. The enzyme is fully functional and retains the preference for NADP+ cofactor from the parent E. coli domain II |
| 1.4.1.2 | co-crystallization of GDH with hexachlorophene and 3-(3,5-dibromo)-4-hydroxybenzylidine-5-iodo-1,3-dihydro-indol-2-one is performed using the hanging drop, vapor-diffusion method at room temperature. In both cases, the drops are formed using a 1:1 mix of protein and reservoir solutions |
| 1.4.1.2 | crystal structure determination and analysis |
| 1.4.1.2 | crystallized in the apo- and holoenzyme forms. Crystals are obtained using 2-propanol and polyethylene glycol MME 550 as precipitants for the apoenzyme and holoenzyme, respectively. The apoenzyme crystals belong to the trigonal space group P3(1)21 or its enantiomorph P3(2)21. The holoenzyme crystals belong to the orthorhombic space group P2(1)2(1)2(1) |
| 1.4.1.2 | native apo-enzyme, poor quality of native crystals is resolved by derivatization with selenomethionine, X-ray diffraction structure determination and analysis at 2.94 A resolution, single-wavelength anomalous diffraction methods |
| 1.4.1.2 | purified recombinant AtGDH1 in the apo-form and in complex with NAD+, X-ray diffraction structure determination and analysis at 2.59 and 2.03 A resolution, respectively. Most of the subunits in the crystal structures, including those in NAD+ complex, are in open conformation, with domain II forming a large (albeit variable) angle with domain I. One of the subunits of the AtGDH1-NAD+ hexamer contains a serendipitous 2-oxoglutarate molecule in the active site, causing a dramatic closure of the domains |
| 1.4.1.2 | purified recombinant wild-type and SeMet-labeled GDHs, hanging drop vapour diffusion method, at 20°C, protein in 10 mM Tris-HCl, pH 7.0, mixing of 0.002 ml of protein solution with 0.001 ml of reservoir solution containing 2.0 M ammonium sulfate, 0.1 M sodium cacodylate, pH 6.5, 200 mM NaCl, and equilibration with 0.5 ml reservoir solution, X-ray diffraction structure determination and analysis at 3.5 A resolution |
| 1.4.1.2 | structure of recombinant GDH1 in the apo-form and in complex with NAD+ at 2.59 and 2.03 A resolution, respectively. Both in the apo form and in 1:1 complex with NAD+, it forms D3-symmetric homohexamers. A subunit of GDH1 consists of domain I, which is involved in hexamer formation and substrate binding, and of domain II which binds coenzyme |
