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
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.; J. Biol. Chem. 284, 4796-4805 (2009) 
Data extracted from this reference:
Cloned(Commentary)
Commentary
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 (nomen rejiciendum)
Engineering
Amino acid exchange
Commentary
Organism
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
Metals/Ions
Commentary
Organism
Structure
Fe-S center
-
Brucella melitensis
Fe-S center
-
Pseudomonas denitrificans (nomen rejiciendum)
Molecular Weight [Da]
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
50000
-
SDS-PAGE, purified CobG protein
Brucella melitensis
50000
-
SDS-PAGE, purified CobG protein
Pseudomonas denitrificans (nomen rejiciendum)
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
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
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Brucella melitensis
Q8YHT1
-
-
Brucella melitensis 16M
Q8YHT1
-
-
Pseudomonas denitrificans (nomen rejiciendum)
-
-
-
Purification (Commentary)
Commentary
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
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)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
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
-
-
-
?
Cloned(Commentary) (protein specific)
Commentary
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 (nomen rejiciendum)
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
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)
Metals/Ions
Commentary
Organism
Structure
Fe-S center
-
Brucella melitensis
Fe-S center
-
Pseudomonas denitrificans (nomen rejiciendum)
Molecular Weight [Da] (protein specific)
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
50000
-
SDS-PAGE, purified CobG protein
Brucella melitensis
50000
-
SDS-PAGE, purified CobG protein
Pseudomonas denitrificans (nomen rejiciendum)
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
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)
Commentary
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
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)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
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
General Information
Commentary
Organism
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)
General Information
Commentary
Organism
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
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
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
2
3
-
3
-
-
2
-
-
-
-
-
3
-
-
-
-
-
-
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-
-
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-
2
-
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14
-
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-
-
-
-
2
2
3
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-
-
2
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
2
2
-
-
-
686660
Iida
Mechanism of the ring contract ...
Pseudomonas denitrificans (nomen rejiciendum)
FEBS J.
274
3475-3481
2007
-
-
-
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2
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1
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-
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3
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1
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1
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2
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3
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671939
Heldt
Aerobic synthesis of vitamin B ...
Rhodobacter capsulatus
Biochem. Soc. Trans.
33
815-819
2005
-
-
-
-
-
-
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1
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1
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1
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1
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1
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1
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1
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1
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2
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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
-
-
1
-
-
-
-
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1
1
2
-
2
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1
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4
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1
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1
1
2
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1
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4
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-
-
-
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-
-
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-
3209
Roessner
Overexpression in Escherichia ...
Pseudomonas denitrificans (nomen rejiciendum)
Protein Expr. Purif.
6
155-163
1995
-
-
1
-
-
-
-
-
-
1
2
1
-
1
-
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1
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2
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1
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1
1
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1
2
1
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1
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2
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15269
Scott
Biosynthesis of vitamin B12. D ...
Pseudomonas denitrificans (nomen rejiciendum)
FEBS Lett.
331
105-108
1993
-
-
1
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-
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2
1
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1
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1
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2
1
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2
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-
-
-
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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
-
-
1
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1
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1
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4
-
2
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1
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1
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6
-
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1
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1
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1
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4
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1
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1
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6
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