Mechanism of the 6-hydroxy-3-succinoyl-pyridine 3-monooxygenase flavoprotein from Pseudomonas putida S16
Yu, H.; Hausinger, R.; Tang, H.; Xu, P.; J. Biol. Chem. 289, 29158-29170 (2014) 
Data extracted from this reference:
Cloned(Commentary)
Cloned (Commentary)
Organism
gene hspB, phylogenetic analysis and tree, recombinant expression of His6-tagged enzyme in Escherichia coli strain BL21(DE3)
Pseudomonas putida
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
stopped-flow spectroscopy and kinetic analysis
Pseudomonas putida
0.0294
-
NADH
pH 8.0, 25°C, recombinant His-tagged enzyme
Pseudomonas putida
0.173
-
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate
pH 8.0, 25°C, recombinant His-tagged enzyme
Pseudomonas putida
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
ID
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate + 2 NADH + 2 H+ + O2
Pseudomonas putida
-
2,5-dihydroxypyridine + succinate semialdehyde + 2 NAD+ + H2O
-
-
?
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate + 2 NADH + 2 H+ + O2
Pseudomonas putida S16
-
2,5-dihydroxypyridine + succinate semialdehyde + 2 NAD+ + H2O
-
-
?
Organism
Organism
UniProt
Commentary
Textmining
Pseudomonas putida
-
-
-
Pseudomonas putida S16
-
-
-
Purification (Commentary)
Purification (Commentary)
Organism
recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
Pseudomonas putida
Reaction
Reaction
Commentary
Organism
Reaction ID
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate + 2 NADH + 2 H+ + O2 = 2,5-dihydroxypyridine + succinate semialdehyde + 2 NAD+ + H2O
catalytic mechanism, overview. In contrast to conclusions reported previously, the second product of the HspB reaction is shown to be succinate, with isotope labeling experiments providing direct evidence that the newly introduced oxygen atom of succinate is derived from H2O. Reduced HspB reacts with oxygen to form a C(4a)-(hydro)peroxyflavin intermediate before it is converted to the oxidized flavoenzyme species. The formed C(4a)-hydroperoxyflavin intermediate reacts with HSP to form an intermediate that is hydrolyzed to the products 2,5-dihydroxypyridine and succinate
Pseudomonas putida
Storage Stability
Storage Stability
Organism
-80°C, purified recombinant His-tagged enzyme HspB, as a frozen solution or dry powder, no change in activity for at least 3 months, pH 8.0
Pseudomonas putida
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
Substrate Product ID
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate + 2 NADH + 2 H+ + O2
-
745310
Pseudomonas putida
2,5-dihydroxypyridine + succinate semialdehyde + 2 NAD+ + H2O
-
-
-
?
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate + 2 NADH + 2 H+ + O2
-
745310
Pseudomonas putida S16
2,5-dihydroxypyridine + succinate semialdehyde + 2 NAD+ + H2O
-
-
-
?
additional information
mass spectrometric analysis of substrates and products, with recombinant enzyme
745310
Pseudomonas putida
?
-
-
-
-
additional information
mass spectrometric analysis of substrates and products, with recombinant enzyme
745310
Pseudomonas putida S16
?
-
-
-
-
Synonyms
Synonyms
Commentary
Organism
6-hydroxy-3-succinoyl-pyridine 3-monooxygenase
-
Pseudomonas putida
HSP 3-monooxygenase
-
Pseudomonas putida
hspB
-
Pseudomonas putida
Temperature Optimum [°C]
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
25
-
assay at
Pseudomonas putida
Turnover Number [1/s]
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
5.49
-
NADH
pH 8.0, 25°C, recombinant His-tagged enzyme
Pseudomonas putida
7.74
-
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate
pH 8.0, 25°C, recombinant His-tagged enzyme
Pseudomonas putida
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
8
-
assay at
Pseudomonas putida
Cofactor
Cofactor
Commentary
Organism
Structure
FAD
-
Pseudomonas putida
NADH
-
Pseudomonas putida
Cloned(Commentary) (protein specific)
Commentary
Organism
gene hspB, phylogenetic analysis and tree, recombinant expression of His6-tagged enzyme in Escherichia coli strain BL21(DE3)
Pseudomonas putida
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
FAD
-
Pseudomonas putida
NADH
-
Pseudomonas putida
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
stopped-flow spectroscopy and kinetic analysis
Pseudomonas putida
0.0294
-
NADH
pH 8.0, 25°C, recombinant His-tagged enzyme
Pseudomonas putida
0.173
-
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate
pH 8.0, 25°C, recombinant His-tagged enzyme
Pseudomonas putida
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
ID
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate + 2 NADH + 2 H+ + O2
Pseudomonas putida
-
2,5-dihydroxypyridine + succinate semialdehyde + 2 NAD+ + H2O
-
-
?
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate + 2 NADH + 2 H+ + O2
Pseudomonas putida S16
-
2,5-dihydroxypyridine + succinate semialdehyde + 2 NAD+ + H2O
-
-
?
Purification (Commentary) (protein specific)
Commentary
Organism
recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
Pseudomonas putida
Storage Stability (protein specific)
Storage Stability
Organism
-80°C, purified recombinant His-tagged enzyme HspB, as a frozen solution or dry powder, no change in activity for at least 3 months, pH 8.0
Pseudomonas putida
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ID
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate + 2 NADH + 2 H+ + O2
-
745310
Pseudomonas putida
2,5-dihydroxypyridine + succinate semialdehyde + 2 NAD+ + H2O
-
-
-
?
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate + 2 NADH + 2 H+ + O2
-
745310
Pseudomonas putida S16
2,5-dihydroxypyridine + succinate semialdehyde + 2 NAD+ + H2O
-
-
-
?
additional information
mass spectrometric analysis of substrates and products, with recombinant enzyme
745310
Pseudomonas putida
?
-
-
-
-
additional information
mass spectrometric analysis of substrates and products, with recombinant enzyme
745310
Pseudomonas putida S16
?
-
-
-
-
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
25
-
assay at
Pseudomonas putida
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
5.49
-
NADH
pH 8.0, 25°C, recombinant His-tagged enzyme
Pseudomonas putida
7.74
-
4-(6-hydroxypyridin-3-yl)-4-oxobutanoate
pH 8.0, 25°C, recombinant His-tagged enzyme
Pseudomonas putida
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
8
-
assay at
Pseudomonas putida
General Information
General Information
Commentary
Organism
evolution
phylogenetic analysis reveals that HspB is the most closely related to two p-nitrophenol 4-monooxygenases, and the experimental results exhibit that p-nitrophenol is a substrate of HspB
Pseudomonas putida
additional information
free H2O2 does not catalyze the HspB enzyme reaction
Pseudomonas putida
physiological function
6-hydroxy-3-succinoyl-pyridine (HSP) 3-monooxygenase (HspB) is a flavoprotein essential to the pyrrolidine pathway of nicotine degradation, it catalyzes pyridine-ring beta-hydroxylation, resulting in carbon-carbon cleavage and production of 2,5-dihydroxypyridine
Pseudomonas putida
General Information (protein specific)
General Information
Commentary
Organism
evolution
phylogenetic analysis reveals that HspB is the most closely related to two p-nitrophenol 4-monooxygenases, and the experimental results exhibit that p-nitrophenol is a substrate of HspB
Pseudomonas putida
additional information
free H2O2 does not catalyze the HspB enzyme reaction
Pseudomonas putida
physiological function
6-hydroxy-3-succinoyl-pyridine (HSP) 3-monooxygenase (HspB) is a flavoprotein essential to the pyrrolidine pathway of nicotine degradation, it catalyzes pyridine-ring beta-hydroxylation, resulting in carbon-carbon cleavage and production of 2,5-dihydroxypyridine
Pseudomonas putida
Other publictions for EC 1.14.13.163
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
Synonyms
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)
744640
Wei
Characterization of a novel n ...
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Catalysts
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272-281
2017
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1
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18
2
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1
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1
-
1
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-
1
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-
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2
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3
1
3
1
1
2
2
1
1
1
2
-
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1
1
2
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18
-
2
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1
-
1
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-
1
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-
2
-
3
1
1
1
2
2
1
1
1
-
-
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2
2
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2
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3
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2
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4
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2
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2
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2
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2
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741691
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Molecular mechanism of nicoti ...
Ochrobactrum sp.
Appl. Environ. Microbiol.
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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
1
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3
3
-
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-
745310
Yu
Mechanism of the 6-hydroxy-3- ...
Pseudomonas putida, Pseudomonas putida S16
J. Biol. Chem.
289
29158-29170
2014
-
-
1
-
-
-
-
3
-
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2
-
4
-
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1
1
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-
1
4
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3
1
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2
1
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-
2
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1
2
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-
3
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-
2
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-
1
-
-
-
1
4
-
1
-
-
2
1
-
-
-
-
3
3
-
-
-
746248
Li
6-hydroxy-3-succinoylpyridine ...
Agrobacterium tumefaciens, Agrobacterium tumefaciens S33
PLoS ONE
9
e103324
2014
-
1
1
-
-
-
-
-
-
-
1
2
-
8
-
-
1
-
-
-
2
-
6
1
4
1
-
-
-
1
-
-
2
-
-
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-
1
1
2
-
-
-
-
-
-
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-
1
2
-
-
-
1
-
-
2
-
6
1
1
-
-
-
1
-
-
-
-
2
2
-
-
-
718677
Wang
Identification of nicotine bio ...
Agrobacterium tumefaciens, Agrobacterium tumefaciens S33
Appl. Microbiol. Biotechnol.
95
1567-1578
2012
-
-
-
-
-
-
-
-
-
-
-
-
-
10
-
-
-
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2
-
1
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2
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-
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-
1
1
-
-
-
719982
Tang
A novel NADH-dependent and FAD ...
Pseudomonas putida, Pseudomonas putida S16
J. Biol. Chem.
286
39179-39187
2011
1
-
-
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1
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-
2
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2
2
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9
-
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1
-
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-
1
2
2
1
2
1
-
1
1
1
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1
3
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1
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3
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1
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2
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2
2
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1
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1
2
2
1
1
-
1
1
1
-
1
-
-
1
1
-
-
-
690527
Tang
A novel gene, encoding 6-hydro ...
Pseudomonas putida, Pseudomonas putida S16
Appl. Environ. Microbiol.
74
1567-1574
2008
-
-
1
-
-
-
-
-
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1
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5
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2
1
1
1
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1
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1
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1
1
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1
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2
1
1
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1
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