Data extracted from this reference:
Cloned(Commentary)
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 (nomen rejiciendum)
Engineering
C358A
no activity, no Fe-S center
Brucella melitensis
C358A
no activity, no Fe-S center
Pseudomonas denitrificans (nomen rejiciendum)
C364A
no activity, no Fe-S center
Brucella melitensis
C364A
no activity, no Fe-S center
Pseudomonas denitrificans (nomen rejiciendum)
C394A
no activity, no Fe-S center
Brucella melitensis
C394A
no activity, no Fe-S center
Pseudomonas denitrificans (nomen rejiciendum)
C398A
no activity, no Fe-S center
Brucella melitensis
C398A
no activity, no Fe-S center
Pseudomonas denitrificans (nomen rejiciendum)
C42A
active, Fe-S center present
Brucella melitensis
C42A
active, Fe-S center present
Pseudomonas denitrificans (nomen rejiciendum)
H390A
active, Fe-S center present
Brucella melitensis
H390A
active, Fe-S center present
Pseudomonas denitrificans (nomen rejiciendum)
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
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 (nomen rejiciendum)
Metals/Ions
Fe-S center
Brucella melitensis
Fe-S center
Pseudomonas denitrificans (nomen rejiciendum)
Molecular Weight [Da]
50000
SDS-PAGE, purified CobG protein
Brucella melitensis
50000
SDS-PAGE, purified CobG protein
Pseudomonas denitrificans (nomen rejiciendum)
Natural Substrates/ Products (Substrates)
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 (nomen rejiciendum)
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
Brucella melitensis
Q8YHT1
Brucella melitensis 16M
Q8YHT1
Pseudomonas denitrificans (nomen rejiciendum)
Purification (Commentary)
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
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 (nomen rejiciendum)
Substrates and Products (Substrate)
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
698972
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
698972
Pseudomonas denitrificans (nomen rejiciendum)
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
698972
Brucella melitensis 16M
precorrin-3B + NAD+ + H2O
?
Synonyms
Bmei0715
Brucella melitensis
CobG
Pseudomonas denitrificans (nomen rejiciendum)
precorrin-3A monooxygenase
Brucella melitensis
precorrin-3A monooxygenase
Pseudomonas denitrificans (nomen rejiciendum)
Cloned(Commentary) (protein specific)
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 (nomen rejiciendum)
Engineering (protein specific)
C358A
no activity, no Fe-S center
Brucella melitensis
C358A
no activity, no Fe-S center
Pseudomonas denitrificans (nomen rejiciendum)
C364A
no activity, no Fe-S center
Brucella melitensis
C364A
no activity, no Fe-S center
Pseudomonas denitrificans (nomen rejiciendum)
C394A
no activity, no Fe-S center
Brucella melitensis
C394A
no activity, no Fe-S center
Pseudomonas denitrificans (nomen rejiciendum)
C398A
no activity, no Fe-S center
Brucella melitensis
C398A
no activity, no Fe-S center
Pseudomonas denitrificans (nomen rejiciendum)
C42A
active, Fe-S center present
Brucella melitensis
C42A
active, Fe-S center present
Pseudomonas denitrificans (nomen rejiciendum)
H390A
active, Fe-S center present
Brucella melitensis
H390A
active, Fe-S center present
Pseudomonas denitrificans (nomen rejiciendum)
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
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 (nomen rejiciendum)
Metals/Ions (protein specific)
Fe-S center
Brucella melitensis
Fe-S center
Pseudomonas denitrificans (nomen rejiciendum)
Molecular Weight [Da] (protein specific)
50000
SDS-PAGE, purified CobG protein
Brucella melitensis
50000
SDS-PAGE, purified CobG protein
Pseudomonas denitrificans (nomen rejiciendum)
Natural Substrates/ Products (Substrates) (protein specific)
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 (nomen rejiciendum)
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
?
Purification (Commentary) (protein specific)
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
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 (nomen rejiciendum)
Substrates and Products (Substrate) (protein specific)
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
698972
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
698972
Pseudomonas denitrificans (nomen rejiciendum)
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
698972
Brucella melitensis 16M
precorrin-3B + NAD+ + H2O
?
General Information
metabolism
vitamin B12/cobalamin biosynthesis, the 4Fe-4S center, mononuclear non-heme iron, is necessary for enzyme activity
Brucella melitensis
metabolism
vitamin B12/cobalamin biosynthesis, the 4Fe-4S center, mononuclear non-heme iron, is necessary for enzyme activity
Pseudomonas denitrificans (nomen rejiciendum)
General Information (protein specific)
metabolism
vitamin B12/cobalamin biosynthesis, the 4Fe-4S center, mononuclear non-heme iron, is necessary for enzyme activity
Brucella melitensis
metabolism
vitamin B12/cobalamin biosynthesis, the 4Fe-4S center, mononuclear non-heme iron, is necessary for enzyme activity
Pseudomonas denitrificans (nomen rejiciendum)
Other publictions for EC 1.14.13.83
698972
Schroeder
Demonstration that CobG, the m ...
Brucella melitensis 16M, Brucella melitensis, Pseudomonas denitrificans (nomen rejiciendum)
J. Biol. Chem.
284
4796-4805
2009
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686660
Iida
Mechanism of the ring contract ...
Pseudomonas denitrificans (nomen rejiciendum)
FEBS J.
274
3475-3481
2007
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671939
Heldt
Aerobic synthesis of vitamin B ...
Rhodobacter capsulatus
Biochem. Soc. Trans.
33
815-819
2005
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650818
Stamford
Biosynthesis of vitamin B12: t ...
Pseudomonas denitrificans (nomen rejiciendum), Pseudomonas denitrificans (nomen rejiciendum) G3575
Chem. Biol.
4
445-451
1997
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3209
Roessner
Overexpression in Escherichia ...
Pseudomonas denitrificans (nomen rejiciendum)
Protein Expr. Purif.
6
155-163
1995
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15269
Scott
Biosynthesis of vitamin B12. D ...
Pseudomonas denitrificans (nomen rejiciendum)
FEBS Lett.
331
105-108
1993
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15270
Debussche
Biosynthesis of the corrin mac ...
Pseudomonas denitrificans (nomen rejiciendum), Pseudomonas denitrificans (nomen rejiciendum) SC510 (RifT)
J. Bacteriol.
175
7430-7440
1993
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