Cloned (Comment) | Organism |
---|---|
Brucella melitensis cobG is PCR-amplified, overexpression of recombinant enzyme in Escherichia coli BL21 (DE3) inducible with isopropyl-1-thio-beta-D-galactopyranoside | Brucella melitensis |
Pseudomonas denitrificans cobG is PCR-amplified in plasmid pCR427 and cloned into pET14b, overexpression of recombinant enzyme in Escherichia coli BL21 (DE3) inducible with isopropyl-1-thio-beta-D-galactopyranoside | Pseudomonas denitrificans (nom. rej.) |
Protein Variants | Comment | Organism |
---|---|---|
C358A | no activity, no Fe-S center | Pseudomonas denitrificans (nom. rej.) |
C358A | no activity, no Fe-S center | Brucella melitensis |
C364A | no activity, no Fe-S center | Pseudomonas denitrificans (nom. rej.) |
C364A | no activity, no Fe-S center | Brucella melitensis |
C394A | no activity, no Fe-S center | Pseudomonas denitrificans (nom. rej.) |
C394A | no activity, no Fe-S center | Brucella melitensis |
C398A | no activity, no Fe-S center | Pseudomonas denitrificans (nom. rej.) |
C398A | no activity, no Fe-S center | Brucella melitensis |
C42A | active, Fe-S center present | Pseudomonas denitrificans (nom. rej.) |
C42A | active, Fe-S center present | Brucella melitensis |
H390A | active, Fe-S center present | Pseudomonas denitrificans (nom. rej.) |
H390A | active, Fe-S center present | Brucella melitensis |
additional information | for in vivo enzyme assays an Escherichia coli strain with all necessary genes for the production of cobyric acid except the cobG gene is used for complementation studies in combination with wild-type and mutant cobG genes (site-directed mutagenesis), for in vitro assays an Escherichia coli strain with a coupled multi-enzyme system (plasmid for the production of hydrogenobyrinic acid lacking the cobG gene) is combined with crude extract of an Escherichia coli strain overproducing GobG | Pseudomonas denitrificans (nom. rej.) |
additional information | for in vivo enzyme assays an Escherichia coli strain with all necessary genes for the production of cobyric acid except the cobG gene is used for complementation studies in combination with wild-type and mutant cobG genes (site-directed mutagenesis), for in vitro assays an Escherichia coli strain with a coupled multi-enzyme system (plasmid for the production of hydrogenobyrinic acid lacking the cobG gene) is combined with crude extract of an Escherichia coli strain overproducing GobG | Brucella melitensis |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Fe-S center | - |
Pseudomonas denitrificans (nom. rej.) | |
Fe-S center | - |
Brucella melitensis |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
50000 | - |
SDS-PAGE, purified CobG protein | Pseudomonas denitrificans (nom. rej.) |
50000 | - |
SDS-PAGE, purified CobG protein | Brucella melitensis |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
precorrin-3A + NADH + H+ + O2 | Brucella melitensis | the Brucella melitensis enzyme is fully active in vitro (20 mM Tris-HCl buffer, pH 8.0, with 100 mM NaCl, S-adenosyl-L-methionine (SAM), 5-aminolevulinic acid (ALA) and NADPH, aerobic, dark, 4°C) and the mononuclear non-heme iron is reducible by dithionite and then is able to react with NO as oxygen analogue in the presence of the substrate | precorrin-3B + NAD+ + H2O | - |
? | |
precorrin-3A + NADH + H+ + O2 | Pseudomonas denitrificans (nom. rej.) | the Pseudomonas denitrificans enzyme is active in vivo but inactive in vitro (20 mM Tris-HCl buffer, pH 8.0, with 100 mM NaCl, S-adenosyl-L-methionine (SAM), 5-aminolevulinic acid (ALA) and NADPH, aerobic, dark, 4°C), the mononuclear non-heme iron is not reducible and probably rapidly inactivated outside the bacterium | precorrin-3B + NAD+ + H2O | - |
? | |
precorrin-3A + NADH + H+ + O2 | Brucella melitensis 16M | the Brucella melitensis enzyme is fully active in vitro (20 mM Tris-HCl buffer, pH 8.0, with 100 mM NaCl, S-adenosyl-L-methionine (SAM), 5-aminolevulinic acid (ALA) and NADPH, aerobic, dark, 4°C) and the mononuclear non-heme iron is reducible by dithionite and then is able to react with NO as oxygen analogue in the presence of the substrate | precorrin-3B + NAD+ + H2O | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Brucella melitensis | Q8YHT1 | - |
- |
Brucella melitensis 16M | Q8YHT1 | - |
- |
Pseudomonas denitrificans (nom. rej.) | - |
- |
- |
Purification (Comment) | Organism |
---|---|
centrifugation of cells, pellet resuspended in binding buffer (20 mM Tris-HCl, pH 8.0, containing 0.5 M NaCl and 5 mM imidazole), cells broken up with a cell disrupter or sonicated, centrifugation, supernatant transferred to an anaerobic glove box, applied to a metal affinity chromatography column charged with Ni2+, washed with buffer with 20 mM imidazole, elution with 400 mM imidazole | Pseudomonas denitrificans (nom. rej.) |
centrifugation of cells, pellet resuspended in binding buffer (20 mM Tris-HCl, pH 8.0, containing 0.5 M NaCl and 5 mM imidazole), cells broken up with a cell disrupter or sonicated, centrifugation, supernatant transferred to an anaerobic glove box, applied to a metal affinity chromatography column charged with Ni2+, washed with buffer with 20 mM imidazole, elution with 400 mM imidazole | Brucella melitensis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
precorrin-3A + NADH + H+ + O2 | the Brucella melitensis enzyme is fully active in vitro (20 mM Tris-HCl buffer, pH 8.0, with 100 mM NaCl, S-adenosyl-L-methionine (SAM), 5-aminolevulinic acid (ALA) and NADPH, aerobic, dark, 4°C) and the mononuclear non-heme iron is reducible by dithionite and then is able to react with NO as oxygen analogue in the presence of the substrate | Brucella melitensis | precorrin-3B + NAD+ + H2O | - |
? | |
precorrin-3A + NADH + H+ + O2 | the Pseudomonas denitrificans enzyme is active in vivo but inactive in vitro (20 mM Tris-HCl buffer, pH 8.0, with 100 mM NaCl, S-adenosyl-L-methionine (SAM), 5-aminolevulinic acid (ALA) and NADPH, aerobic, dark, 4°C), the mononuclear non-heme iron is not reducible and probably rapidly inactivated outside the bacterium | Pseudomonas denitrificans (nom. rej.) | precorrin-3B + NAD+ + H2O | - |
? | |
precorrin-3A + NADH + H+ + O2 | the Brucella melitensis enzyme is fully active in vitro (20 mM Tris-HCl buffer, pH 8.0, with 100 mM NaCl, S-adenosyl-L-methionine (SAM), 5-aminolevulinic acid (ALA) and NADPH, aerobic, dark, 4°C) and the mononuclear non-heme iron is reducible by dithionite and then is able to react with NO as oxygen analogue in the presence of the substrate | Brucella melitensis 16M | precorrin-3B + NAD+ + H2O | - |
? |
Synonyms | Comment | Organism |
---|---|---|
Bmei0715 | - |
Brucella melitensis |
CobG | - |
Pseudomonas denitrificans (nom. rej.) |
CobG | - |
Brucella melitensis |
precorrin-3A monooxygenase | - |
Pseudomonas denitrificans (nom. rej.) |
precorrin-3A monooxygenase | - |
Brucella melitensis |
General Information | Comment | Organism |
---|---|---|
metabolism | vitamin B12/cobalamin biosynthesis, the 4Fe-4S center, mononuclear non-heme iron, is necessary for enzyme activity | Pseudomonas denitrificans (nom. rej.) |
metabolism | vitamin B12/cobalamin biosynthesis, the 4Fe-4S center, mononuclear non-heme iron, is necessary for enzyme activity | Brucella melitensis |