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Literature summary extracted from

  • Schroeder, S.; Lawrence, A.D.; Biedendieck, R.; Rose, R.S.; Deery, E.; Graham, R.M.; McLean, K.J.; Munro, A.W.; Rigby, S.E.; Warren, M.J.
    Demonstration that CobG, the monooxygenase associated with the ring contraction process of the aerobic cobalamin (vitamin B12) biosynthetic pathway, contains an Fe-S center and a mononuclear non-heme iron center (2009), J. Biol. Chem., 284, 4796-4805.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.13.83 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
1.14.13.83 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

EC Number Protein Variants Comment Organism
1.14.13.83 C358A no activity, no Fe-S center Pseudomonas denitrificans (nom. rej.)
1.14.13.83 C358A no activity, no Fe-S center Brucella melitensis
1.14.13.83 C364A no activity, no Fe-S center Pseudomonas denitrificans (nom. rej.)
1.14.13.83 C364A no activity, no Fe-S center Brucella melitensis
1.14.13.83 C394A no activity, no Fe-S center Pseudomonas denitrificans (nom. rej.)
1.14.13.83 C394A no activity, no Fe-S center Brucella melitensis
1.14.13.83 C398A no activity, no Fe-S center Pseudomonas denitrificans (nom. rej.)
1.14.13.83 C398A no activity, no Fe-S center Brucella melitensis
1.14.13.83 C42A active, Fe-S center present Pseudomonas denitrificans (nom. rej.)
1.14.13.83 C42A active, Fe-S center present Brucella melitensis
1.14.13.83 H390A active, Fe-S center present Pseudomonas denitrificans (nom. rej.)
1.14.13.83 H390A active, Fe-S center present Brucella melitensis
1.14.13.83 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.)
1.14.13.83 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

EC Number Metals/Ions Comment Organism Structure
1.14.13.83 Fe-S center
-
 Pseudomonas denitrificans (nom. rej.)
1.14.13.83 Fe-S center
-
 Brucella melitensis

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.14.13.83 50000
-
 SDS-PAGE, purified CobG protein Pseudomonas denitrificans (nom. rej.)
1.14.13.83 50000
-
 SDS-PAGE, purified CobG protein Brucella melitensis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.13.83 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
-
 ?
1.14.13.83 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
-
 ?
1.14.13.83 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

EC Number Organism UniProt Comment Textmining
1.14.13.83 Brucella melitensis Q8YHT1
-
-
1.14.13.83 Brucella melitensis 16M Q8YHT1
-
-
1.14.13.83 Pseudomonas denitrificans (nom. rej.)
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.13.83 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.)
1.14.13.83 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 and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.13.83 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
-
 ?
1.14.13.83 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
-
 ?
1.14.13.83 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

EC Number Synonyms Comment Organism
1.14.13.83 Bmei0715
-
 Brucella melitensis
1.14.13.83 CobG
-
 Pseudomonas denitrificans (nom. rej.)
1.14.13.83 CobG
-
 Brucella melitensis
1.14.13.83 precorrin-3A monooxygenase
-
 Pseudomonas denitrificans (nom. rej.)
1.14.13.83 precorrin-3A monooxygenase
-
 Brucella melitensis

General Information

EC Number General Information Comment Organism
1.14.13.83 metabolism vitamin B12/cobalamin biosynthesis, the 4Fe-4S center, mononuclear non-heme iron, is necessary for enzyme activity Pseudomonas denitrificans (nom. rej.)
1.14.13.83 metabolism vitamin B12/cobalamin biosynthesis, the 4Fe-4S center, mononuclear non-heme iron, is necessary for enzyme activity Brucella melitensis