BRENDA - Enzyme Database
show all sequences of 1.7.1.B1

Subfunctionality of hydride transferases of the old yellow enzyme family of flavoproteins of Pseudomonas putida

Van Dillewijn, P.; Wittich, R.M.; Caballero, A.; Ramos, J.L.; Appl. Environ. Microbiol. 74, 6703-6708 (2008)

Data extracted from this reference:

Cloned(Commentary)
Cloned (Commentary)
Organism
xenB gene is amplified using appropriate primers with BamHI and HindIII sites and Pseudomonas putida KT2440 chromosomal DNA as a template. After digestion with restriction enzymes, the PCR product is ligated into the pET28b(+) vector. Resulting plasmid contains the coding sequence in frame with a DNA sequence encoding a His-tag, which resulted in a hexahistidine tail. For protein-His6 expression, plasmid is transformed into Escherichia coli BL21.
Pseudomonas putida
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.027
-
glycerol trinitrate
commentary
Pseudomonas putida
0.037
-
2,4,6-trinitrotoluene
-
Pseudomonas putida
0.044
-
N-ethylmaleimide
commentary
Pseudomonas putida
0.077
-
NADPH
-
Pseudomonas putida
0.158
-
cyclohexenone
commentary
Pseudomonas putida
Molecular Weight [Da]
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
additional information
-
determined by gel filtration, molecular masses of the His6-tagged proteins XenA to XenF are in the range of 40900 to 42600 Da
Pseudomonas putida
Organism
Organism
UniProt
Commentary
Textmining
Pseudomonas putida
Q88PD0
-
-
Pseudomonas putida KT 2240
Q88PD0
-
-
Purification (Commentary)
Purification (Commentary)
Organism
His6-tagged protein is purified by nickel affinity chromatography and eluted with a continuous imidazole gradient.
Pseudomonas putida
Reaction
Reaction
Commentary
Organism
Reaction ID
2,4,6-trinitrotoluene + 2 NADPH + 2 H+ = N-hydroxy-2-methyl-3,5-dinitroaniline + 2 NADP+ + H2O
The amount of N-hydroxy-2-methyl-3,5-dinitroaniline is larger than the amount of N-hydroxy-4-methyl-3,5-dinitroaniline formed. In a further nonenzymatic reaction nitrite is released and N-(2-methyl-3,5-dinitrophenyl)-N-4-methyl-3,5-dinitrophenyl-hydroxylamine and eventually N-(2-methyl-3,5-dinitrophenyl)-N-4-methyl-3,5-dinitroaniline are produced. In adidtion the aromatic ring of 2,4,6-trinitrotoluene is susceptible to nucleophilic attack by hydride ions to form Meisenheimer complex intermediates (monohydride and dihydride complexes) which also form the secondary diaryl hydroxylamines and the secondary diarylamine with release of nitrite
Pseudomonas putida
2,4,6-trinitrotoluene + 2 NADPH + 2 H+ = N-hydroxy-4-methyl-3,5-dinitroaniline + 2 NADP+ + H2O
The amount of N-hydroxy-4-methyl-3,5-dinitroaniline is larger than the amount of N-hydroxy-2-methyl-3,5-dinitroaniline formed. In a further nonenzymatic reaction nitrite is released and N,N-bis-(3,5-dinitrotolyl)-hydroxylamine and eventually N,N-bis-(3,5-dinitrotolyl)-amine are produced. In addition the aromatic ring of 2,4,6-trinitrotoluene is susceptible to nucleophilic attack by hydride ions to form Meisenheimer complex intermediates (monohydride and dihydride complexes) which also form the secondary diaryl hydroxylamines and the secondary diarylamine with release of nitrite
Pseudomonas putida
Specific Activity [micromol/min/mg]
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
additional information
-
Vmax is denoted with 112 micromol/min/mg protein, XenB exhibits the highest Vmax values and the most favorable Vmax/Km relationship for 2,4,6-trinitrotoluene compared to those of the other active xenobiotic reductases of Pseudomonas putida KT2440
Pseudomonas putida
1.88
-
highest specific activity within xenobiotic reductases A to F
Pseudomonas putida
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
Substrate Product ID
2,4,6-trinitrotoluene + 2 NADPH + 2 H+
-
690550
Pseudomonas putida
N-hydroxy-2-methyl-3,5-dinitroaniline + 2 NADP+ + H2O
i.e. 2-hydroxylamino-2,6-dinitrotoluene. The amount of N-hydroxy-2-methyl-3,5-dinitroaniline is larger than the amount of N-hydroxy-4-methyl-3,5-dinitroaniline formed. In a further nonenzymatic reaction nitrite is released and N-(2-methyl-3,5-dinitrophenyl)-N-4-methyl-3,5-dinitrophenyl-hydroxylamine and eventually N-(2-methyl-3,5-dinitrophenyl)-N-4-methyl-3,5-dinitroaniline are produced. In addition the aromatic ring of 2,4,6-trinitrotoluene is susceptibe to nucleophilic attack by hydride ions to form Meisenheimer complex intermediates (monohydride and dihydride complexes) which also form secondary diaryl hydroxylamines and secondary diarylamine with release of nitrite
-
-
?
2,4,6-trinitrotoluene + 2 NADPH + 2 H+
-
690550
Pseudomonas putida KT 2240
N-hydroxy-2-methyl-3,5-dinitroaniline + 2 NADP+ + H2O
i.e. 2-hydroxylamino-2,6-dinitrotoluene. The amount of N-hydroxy-2-methyl-3,5-dinitroaniline is larger than the amount of N-hydroxy-4-methyl-3,5-dinitroaniline formed. In a further nonenzymatic reaction nitrite is released and N-(2-methyl-3,5-dinitrophenyl)-N-4-methyl-3,5-dinitrophenyl-hydroxylamine and eventually N-(2-methyl-3,5-dinitrophenyl)-N-4-methyl-3,5-dinitroaniline are produced. In addition the aromatic ring of 2,4,6-trinitrotoluene is susceptibe to nucleophilic attack by hydride ions to form Meisenheimer complex intermediates (monohydride and dihydride complexes) which also form secondary diaryl hydroxylamines and secondary diarylamine with release of nitrite
-
-
?
2,4,6-trinitrotoluene + 2 NADPH + 2 H+
-
690550
Pseudomonas putida
N-hydroxy-4-methyl-3,5-dinitroaniline + 2 NADP+ + H2O
i.e. 4-hydroxylamino-2,6-dinitrotoluene. The amount of N-hydroxy-4-methyl-3,5-dinitroaniline is larger than the amount of N-hydroxy-2-methyl-3,5-dinitroaniline formed. In a further nonenzymatic reaction nitrite is released and N,N-bis-(3,5-dinitrotolyl)-hydroxylamine and eventually N,N-bis-(3,5-dinitrotolyl)-amine are produced. In addition the aromatic ring of 2,4,6-trinitrotoluene is susceptible to nucleophilic attack by hydride ions to form Meisenheimer complex intermediates (monohydride and dihydride complexes) which also form the secondary diaryl hydroxylamines and the secondary diarylamine with release of nitrite
-
-
?
2,4,6-trinitrotoluene + 2 NADPH + 2 H+
-
690550
Pseudomonas putida KT 2240
N-hydroxy-4-methyl-3,5-dinitroaniline + 2 NADP+ + H2O
i.e. 4-hydroxylamino-2,6-dinitrotoluene. The amount of N-hydroxy-4-methyl-3,5-dinitroaniline is larger than the amount of N-hydroxy-2-methyl-3,5-dinitroaniline formed. In a further nonenzymatic reaction nitrite is released and N,N-bis-(3,5-dinitrotolyl)-hydroxylamine and eventually N,N-bis-(3,5-dinitrotolyl)-amine are produced. In addition the aromatic ring of 2,4,6-trinitrotoluene is susceptible to nucleophilic attack by hydride ions to form Meisenheimer complex intermediates (monohydride and dihydride complexes) which also form the secondary diaryl hydroxylamines and the secondary diarylamine with release of nitrite
-
-
?
cyclohexenone + NADPH + H+
-
690550
Pseudomonas putida
?
-
-
-
?
cyclohexenone + NADPH + H+
-
690550
Pseudomonas putida KT 2240
?
-
-
-
?
glycerol trinitrate + NADPH + H+
-
690550
Pseudomonas putida
?
-
-
-
?
glycerol trinitrate + NADPH + H+
-
690550
Pseudomonas putida KT 2240
?
-
-
-
?
additional information
ability to reduce nitroaromatic compounds, enzyme demonstrates type I and type II hydride transferase activity and reduced the nitro groups of 2,4,6-trinitrotoluene to hydroxylaminodinitrotoluene derivatives. The condensations of the primary products of type I and type II hydride transferases react with each other to yield diarylamines and nitrite, the latter can be further reduced to ammonium and serves as a nitrogen source for microorganisms in vivo
690550
Pseudomonas putida
?
-
-
-
-
additional information
ability to reduce nitroaromatic compounds, enzyme demonstrates type I and type II hydride transferase activity and reduced the nitro groups of 2,4,6-trinitrotoluene to hydroxylaminodinitrotoluene derivatives. The condensations of the primary products of type I and type II hydride transferases react with each other to yield diarylamines and nitrite, the latter can be further reduced to ammonium and serves as a nitrogen source for microorganisms in vivo
690550
Pseudomonas putida KT 2240
?
-
-
-
-
N-ethylmaleimide + NADPH + H+
-
690550
Pseudomonas putida
?
-
-
-
?
Subunits
Subunits
Commentary
Organism
monomer
in solution
Pseudomonas putida
Synonyms
Synonyms
Commentary
Organism
old yellow enzyme
-
Pseudomonas putida
OYE
-
Pseudomonas putida
XenB
-
Pseudomonas putida
xenobiotic reductase B
family of flavoproteins
Pseudomonas putida
Temperature Optimum [°C]
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
25
-
assay at
Pseudomonas putida
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7
-
assay at
Pseudomonas putida
Cofactor
Cofactor
Commentary
Organism
Structure
FMN
in the primary sequence of these proteins, a number of residues involved in interactions with the FMN cofactor is found
Pseudomonas putida
NAD(P)H
enzyme preferentially uses NADPH as a cofactor
Pseudomonas putida
Cloned(Commentary) (protein specific)
Commentary
Organism
xenB gene is amplified using appropriate primers with BamHI and HindIII sites and Pseudomonas putida KT2440 chromosomal DNA as a template. After digestion with restriction enzymes, the PCR product is ligated into the pET28b(+) vector. Resulting plasmid contains the coding sequence in frame with a DNA sequence encoding a His-tag, which resulted in a hexahistidine tail. For protein-His6 expression, plasmid is transformed into Escherichia coli BL21.
Pseudomonas putida
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
FMN
in the primary sequence of these proteins, a number of residues involved in interactions with the FMN cofactor is found
Pseudomonas putida
NAD(P)H
enzyme preferentially uses NADPH as a cofactor
Pseudomonas putida
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.027
-
glycerol trinitrate
commentary
Pseudomonas putida
0.037
-
2,4,6-trinitrotoluene
-
Pseudomonas putida
0.044
-
N-ethylmaleimide
commentary
Pseudomonas putida
0.077
-
NADPH
-
Pseudomonas putida
0.158
-
cyclohexenone
commentary
Pseudomonas putida
Molecular Weight [Da] (protein specific)
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
additional information
-
determined by gel filtration, molecular masses of the His6-tagged proteins XenA to XenF are in the range of 40900 to 42600 Da
Pseudomonas putida
Purification (Commentary) (protein specific)
Commentary
Organism
His6-tagged protein is purified by nickel affinity chromatography and eluted with a continuous imidazole gradient.
Pseudomonas putida
Specific Activity [micromol/min/mg] (protein specific)
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
additional information
-
Vmax is denoted with 112 micromol/min/mg protein, XenB exhibits the highest Vmax values and the most favorable Vmax/Km relationship for 2,4,6-trinitrotoluene compared to those of the other active xenobiotic reductases of Pseudomonas putida KT2440
Pseudomonas putida
1.88
-
highest specific activity within xenobiotic reductases A to F
Pseudomonas putida
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ID
2,4,6-trinitrotoluene + 2 NADPH + 2 H+
-
690550
Pseudomonas putida
N-hydroxy-2-methyl-3,5-dinitroaniline + 2 NADP+ + H2O
i.e. 2-hydroxylamino-2,6-dinitrotoluene. The amount of N-hydroxy-2-methyl-3,5-dinitroaniline is larger than the amount of N-hydroxy-4-methyl-3,5-dinitroaniline formed. In a further nonenzymatic reaction nitrite is released and N-(2-methyl-3,5-dinitrophenyl)-N-4-methyl-3,5-dinitrophenyl-hydroxylamine and eventually N-(2-methyl-3,5-dinitrophenyl)-N-4-methyl-3,5-dinitroaniline are produced. In addition the aromatic ring of 2,4,6-trinitrotoluene is susceptibe to nucleophilic attack by hydride ions to form Meisenheimer complex intermediates (monohydride and dihydride complexes) which also form secondary diaryl hydroxylamines and secondary diarylamine with release of nitrite
-
-
?
2,4,6-trinitrotoluene + 2 NADPH + 2 H+
-
690550
Pseudomonas putida KT 2240
N-hydroxy-2-methyl-3,5-dinitroaniline + 2 NADP+ + H2O
i.e. 2-hydroxylamino-2,6-dinitrotoluene. The amount of N-hydroxy-2-methyl-3,5-dinitroaniline is larger than the amount of N-hydroxy-4-methyl-3,5-dinitroaniline formed. In a further nonenzymatic reaction nitrite is released and N-(2-methyl-3,5-dinitrophenyl)-N-4-methyl-3,5-dinitrophenyl-hydroxylamine and eventually N-(2-methyl-3,5-dinitrophenyl)-N-4-methyl-3,5-dinitroaniline are produced. In addition the aromatic ring of 2,4,6-trinitrotoluene is susceptibe to nucleophilic attack by hydride ions to form Meisenheimer complex intermediates (monohydride and dihydride complexes) which also form secondary diaryl hydroxylamines and secondary diarylamine with release of nitrite
-
-
?
2,4,6-trinitrotoluene + 2 NADPH + 2 H+
-
690550
Pseudomonas putida
N-hydroxy-4-methyl-3,5-dinitroaniline + 2 NADP+ + H2O
i.e. 4-hydroxylamino-2,6-dinitrotoluene. The amount of N-hydroxy-4-methyl-3,5-dinitroaniline is larger than the amount of N-hydroxy-2-methyl-3,5-dinitroaniline formed. In a further nonenzymatic reaction nitrite is released and N,N-bis-(3,5-dinitrotolyl)-hydroxylamine and eventually N,N-bis-(3,5-dinitrotolyl)-amine are produced. In addition the aromatic ring of 2,4,6-trinitrotoluene is susceptible to nucleophilic attack by hydride ions to form Meisenheimer complex intermediates (monohydride and dihydride complexes) which also form the secondary diaryl hydroxylamines and the secondary diarylamine with release of nitrite
-
-
?
2,4,6-trinitrotoluene + 2 NADPH + 2 H+
-
690550
Pseudomonas putida KT 2240
N-hydroxy-4-methyl-3,5-dinitroaniline + 2 NADP+ + H2O
i.e. 4-hydroxylamino-2,6-dinitrotoluene. The amount of N-hydroxy-4-methyl-3,5-dinitroaniline is larger than the amount of N-hydroxy-2-methyl-3,5-dinitroaniline formed. In a further nonenzymatic reaction nitrite is released and N,N-bis-(3,5-dinitrotolyl)-hydroxylamine and eventually N,N-bis-(3,5-dinitrotolyl)-amine are produced. In addition the aromatic ring of 2,4,6-trinitrotoluene is susceptible to nucleophilic attack by hydride ions to form Meisenheimer complex intermediates (monohydride and dihydride complexes) which also form the secondary diaryl hydroxylamines and the secondary diarylamine with release of nitrite
-
-
?
cyclohexenone + NADPH + H+
-
690550
Pseudomonas putida
?
-
-
-
?
cyclohexenone + NADPH + H+
-
690550
Pseudomonas putida KT 2240
?
-
-
-
?
glycerol trinitrate + NADPH + H+
-
690550
Pseudomonas putida
?
-
-
-
?
glycerol trinitrate + NADPH + H+
-
690550
Pseudomonas putida KT 2240
?
-
-
-
?
additional information
ability to reduce nitroaromatic compounds, enzyme demonstrates type I and type II hydride transferase activity and reduced the nitro groups of 2,4,6-trinitrotoluene to hydroxylaminodinitrotoluene derivatives. The condensations of the primary products of type I and type II hydride transferases react with each other to yield diarylamines and nitrite, the latter can be further reduced to ammonium and serves as a nitrogen source for microorganisms in vivo
690550
Pseudomonas putida
?
-
-
-
-
additional information
ability to reduce nitroaromatic compounds, enzyme demonstrates type I and type II hydride transferase activity and reduced the nitro groups of 2,4,6-trinitrotoluene to hydroxylaminodinitrotoluene derivatives. The condensations of the primary products of type I and type II hydride transferases react with each other to yield diarylamines and nitrite, the latter can be further reduced to ammonium and serves as a nitrogen source for microorganisms in vivo
690550
Pseudomonas putida KT 2240
?
-
-
-
-
N-ethylmaleimide + NADPH + H+
-
690550
Pseudomonas putida
?
-
-
-
?
Subunits (protein specific)
Subunits
Commentary
Organism
monomer
in solution
Pseudomonas putida
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
25
-
assay at
Pseudomonas putida
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7
-
assay at
Pseudomonas putida
Other publictions for EC 1.7.1.B1
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)
723954
Fetar
mexEF-oprN multidrug efflux op ...
Pseudomonas aeruginosa
Antimicrob. Agents Chemother.
55
508-514
2011
-
-
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-
-
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1
-
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1
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-
-
-
-
-
-
-
-
-
2
-
-
2
-
-
690550
Van Dillewijn
Subfunctionality of hydride tr ...
Pseudomonas putida, Pseudomonas putida KT 2240
Appl. Environ. Microbiol.
74
6703-6708
2008
-
-
1
-
-
-
-
5
-
-
1
-
-
3
-
-
1
2
-
-
2
-
11
1
4
1
-
-
-
1
-
-
2
-
-
-
-
-
1
2
-
-
-
-
-
-
5
-
-
1
-
-
-
-
1
-
-
2
-
11
1
1
-
-
-
1
-
-
-
-
-
-
-
-
-
690551
Van Dillewijn
Type II hydride transferases f ...
Pseudomonas putida, Pseudomonas putida KT 2240
Appl. Environ. Microbiol.
74
6820-6823
2008
-
-
-
-
-
-
-
2
-
-
-
-
-
3
-
-
1
2
-
-
1
-
4
-
2
1
-
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1
-
-
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-
-
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-
-
-
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-
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2
-
-
-
-
-
-
-
1
-
-
1
-
4
-
1
-
-
-
1
-
-
-
-
-
-
-
-
-
692112
Wittich
OYE flavoprotein reductases in ...
Pseudomonas putida, Pseudomonas putida JLR11
Environ. Sci. Technol.
42
734-739
2008
-
-
1
-
-
-
-
-
-
2
-
-
-
3
-
-
1
2
-
-
-
-
-
-
3
1
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1
-
-
-
-
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-
1
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-
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-
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2
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1
-
-
-
-
-
-
1
-
-
-
1
-
-
-
-
-
-
-
-
-
740929
Ho
-
Characterization of nitroreduc ...
Pseudomonas sp.
Korean J. Microbiol. Biotechnol.
32
230-237
2004
-
-
-
-
-
-
5
-
-
-
1
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-
1
-
-
1
-
-
-
1
-
2
1
-
1
-
-
-
1
-
-
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-
-
-
-
-
-
-
-
-
-
-
5
-
-
-
-
1
-
-
-
-
1
-
-
1
-
2
1
1
-
-
-
1
-
-
-
-
-
-
-
-
-