Cloned (Comment) | Organism |
---|---|
gene MJ1311, recombinant expression in Escherichia coli in inclusion bodies | Methanocaldococcus jannaschii |
Crystallization (Comment) | Organism |
---|---|
purified enzyme, sitting drop vapour diffusion method, mixing of 0.001 ml of 10.3 mg/ml protein in 10 mM potassium phosphate, pH 7.0, with 0.001 ml of reservoir solution composed of 0.2 M ammonium acetate, 30% isopropanol, 0.1 M Tris-HCl, pH 7.5, and equilibration against 0.1 ml reservoir solution, 3-7 days, 20°C, X-ray diffraction structure determination and analysis at 2.64 A resolution, heavy metal labeleing, single isomorphous replacement with anomalous scattering, and structure molecular modeling | Methanocaldococcus jannaschii |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mn2+ | four Mn2+ ions per enzyme homodimer, structure modeling, overview | Methanocaldococcus jannaschii |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
60000 | - |
recombinant enzyme, gel filtration | Methanocaldococcus jannaschii |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Methanocaldococcus jannaschii | Q58707 | - |
- |
Methanocaldococcus jannaschii ATCC 43067 / 43067D / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440 | Q58707 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant solublized and refolded enzyme expressed in Escherichia coli by dialysis and gel filtration | Methanocaldococcus jannaschii |
Renatured (Comment) | Organism |
---|---|
recombinant enzyme from Escherichia coli inclusion bodies by treatment with 20 ml 10 mM Tris-HCl buffer, pH 7.5, containing 1.0 mM EDTA, 4% Triton X-100, incubation at room temperature for 30 min, twice. Then the inclusion bodies are treated with denaturant solution containing 50 mM Tris-HCl buffer, pH 7.5, containing 6 M guanidine-HCl, 0.2 M NaCl, 1 mM EDTA overnight at 4°C. The solubilized enzyme (100 mg protein in 100 ml solution) is gently dropped into 1 l of refolding buffer containing 0.1 M Tris-HCl, pH 7.5, 1 mM MnCl2, and 0.4 M L-arginine, and incubated for 16 h at 4°C, followed by ultrafiltration | Methanocaldococcus jannaschii |
Subunits | Comment | Organism |
---|---|---|
homodimer | 2 x 30000, recombinant enzyme, SDS-PAGE | Methanocaldococcus jannaschii |
More | the asymmetric unit contained two homologous subunits, and the dimer is generated by twofold symmetry. In MJ1311p, Glu125, Leu126 and Trp127 from one subunit are located over the metal-ion-binding site of the other subunit and contribute to the active site, narrowing the substrate-binding cleft. Three-dimensional structural analysis of MJ1311p, overview. The enzyme MJ1311p monomer is folded into an (alpha/beta)8 barrel carrying four additional helical segments, alpha1', alpha2', alpha4', and alpha6', which are inserted before alpha1, alpha2, alpha4, and alpha6, respectively. The quaternary-structural arrangement of MJ1311p is notably different from those of D-TE family enzymes | Methanocaldococcus jannaschii |
Synonyms | Comment | Organism |
---|---|---|
MJ1311 | - |
Methanocaldococcus jannaschii |
MJ1311p | - |
Methanocaldococcus jannaschii |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme belongs to the D-tagatose 3-epimerase (D-TE) family enzymes. The overall fold of the subunit proves to be similar to those of the D-tagatose 3-epimerase from Pseudomonas cichorii and the D-psicose 3-epimerases from Agrobacterium tumefaciens and Clostridium cellulolyticum. But the situation at the subunit-subunit interface differs substantially from that in D-tagatose 3-epimerase family enzymes. In MJ1311p, Glu125, Leu126 and Trp127 from one subunit are located over the metal-ion-binding site of the other subunit and contribute to the active site, narrowing the substrate-binding cleft. Moreover, the nine residues comprising a trinuclear zinc centre in endonuclease IV are strictly conserved in MJ1311p, although a distinct groove involved in DNA binding is not present. The active-site architecture of MJ1311p is quite unique and is substantially different from those of D-tagatose 3-epimerase family enzymes and endonuclease IV | Methanocaldococcus jannaschii |