BRENDA - Reference to 1.14.13.83; Id = 698972

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)

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)

Fe-S center

-

Brucella melitensis

Fe-S center

-

Pseudomonas denitrificans (nomen rejiciendum)

50000

-

SDS-PAGE, purified CobG protein

Brucella melitensis

50000

-

SDS-PAGE, purified CobG protein

Pseudomonas denitrificans (nomen rejiciendum)

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

-

?

Brucella melitensis

Q8YHT1

-

Brucella melitensis 16M

Q8YHT1

-

Pseudomonas denitrificans (nomen rejiciendum)

-

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)

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

-

?

Bmei0715

-

Brucella melitensis

CobG

-

Brucella melitensis

CobG

-

Pseudomonas denitrificans (nomen rejiciendum)

precorrin-3A monooxygenase

-

Brucella melitensis

precorrin-3A monooxygenase

-

Pseudomonas denitrificans (nomen rejiciendum)

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)

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)

Fe-S center

-

Brucella melitensis

Fe-S center

-

Pseudomonas denitrificans (nomen rejiciendum)

50000

-

SDS-PAGE, purified CobG protein

Brucella melitensis

50000

-

SDS-PAGE, purified CobG protein

Pseudomonas denitrificans (nomen rejiciendum)

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

-

?

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)

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

-

?

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)

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)

698972

Schroeder

Demonstration that CobG, the m ...

Brucella melitensis 16M, Brucella melitensis, Pseudomonas denitrificans (nomen rejiciendum)

J. Biol. Chem.

284

4796-4805

2009

-

2

-

14

-

2

3

-

3

-

2

-

3

-

5

-

2

-

14

-

2

3

-

2

-

3

-

2

-

686660

Iida

Mechanism of the ring contract ...

Pseudomonas denitrificans (nomen rejiciendum)

FEBS J.

274

3475-3481

2007

-

2

-

1

-

3

-

1

-

1

-

2

-

3

-

671939

Heldt

Aerobic synthesis of vitamin B ...

Rhodobacter capsulatus

Biochem. Soc. Trans.

33

815-819

2005

-

1

-

1

-

1

-

1

-

2

-

1

-

1

-

1

-

1

-

1

-

2

-

650818

Stamford

Biosynthesis of vitamin B12: t ...

Pseudomonas denitrificans (nomen rejiciendum), Pseudomonas denitrificans (nomen rejiciendum) G3575

Chem. Biol.

4

445-451

1997

-

1

-

1

2

-

2

-

1

-

4

-

1

-

1

-

1

2

-

1

-

4

-

3209

Roessner

Overexpression in Escherichia ...

Pseudomonas denitrificans (nomen rejiciendum)

Protein Expr. Purif.

6

155-163

1995

-

1

-

1

2

1

-

1

-

1

-

2

-

2

-

1

-

1

-

1

2

1

-

1

-

2

-

15269

Scott

Biosynthesis of vitamin B12. D ...

Pseudomonas denitrificans (nomen rejiciendum)

FEBS Lett.

331

105-108

1993

-

1

-

2

1

-

1

-

2

-

1

-

2

1

-

2

-

15270

Debussche

Biosynthesis of the corrin mac ...

Pseudomonas denitrificans (nomen rejiciendum), Pseudomonas denitrificans (nomen rejiciendum) SC510 (RifT)

J. Bacteriol.

175

7430-7440

1993

-

1

-

1

-

1

-

4

-

2

-

1

-

1

-

6

-

1

-

1

-

1

-

1

-

4

-

1

-

1

-

6

-

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

Data extracted from this reference: