BRENDA - Enzyme Database
show all sequences of 4.2.1.106

Structure and functional characterization of a bile acid 7alpha dehydratase BaiE in secondary bile acid synthesis

Bhowmik, S.; Chiu, H.P.; Jones, D.H.; Chiu, H.J.; Miller, M.D.; Xu, Q.; Farr, C.L.; Ridlon, J.M.; Wells, J.E.; Elsliger, M.A.; Wilson, I.A.; Hylemon, P.B.; Lesley, S.A.; Proteins 84, 316-331 (2016)

Data extracted from this reference:

Crystallization (Commentary)
Crystallization
Organism
-
Clostridium hiranonis
-
Clostridium hylemonae
;
Clostridium scindens
Engineering
Amino acid exchange
Commentary
Organism
D106N
inactive mutant
Clostridium scindens
D35N
inactive mutant
Clostridium scindens
H83N
inactive mutant
Clostridium scindens
R146Q
inactive mutant
Clostridium scindens
Y126F
strongly reduced activity compared to wild type
Clostridium scindens
Y30F
inactive mutant
Clostridium scindens
Y54A
inactive mutant
Clostridium scindens
Y54F
reduced activity compared to wild type
Clostridium scindens
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.2
-
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium scindens
4
-
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
25°C in 20 mM Tris-HCl pH 7.3
Clostridium scindens
9.9
-
7alpha-hydroxy-3-oxochol-4-en-24-oate
25°C in 20 mM Tris-HCl pH 7.3
Clostridium scindens
11
-
7alpha-hydroxy-3-oxochol-4-en-24-oate
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium scindens
11.4
-
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium hylemonae
21
-
7alpha-hydroxy-3-oxochol-4-en-24-oate
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium hylemonae
25
-
7alpha-hydroxy-3-oxochol-4-en-24-oate
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium hiranonis
208
-
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium hiranonis
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
Clostridium hiranonis
-
3-oxochola-4,6-dien-24-oyl-CoA
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
Clostridium hylemonae
-
3-oxochola-4,6-dien-24-oyl-CoA
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
Clostridium scindens
-
3-oxochola-4,6-dien-24-oyl-CoA
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
Clostridium hylemonae DSM 15053
-
3-oxochola-4,6-dien-24-oyl-CoA
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
Clostridium scindens ATCC 35704
-
3-oxochola-4,6-dien-24-oyl-CoA
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
Clostridium scindens VPI 12708
-
3-oxochola-4,6-dien-24-oyl-CoA
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
Clostridium hiranonis DSM 13275
-
3-oxochola-4,6-dien-24-oyl-CoA
-
-
?
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Clostridium hiranonis
Q9RB47
-
-
Clostridium hiranonis DSM 13275
Q9RB47
-
-
Clostridium hylemonae
B4YSU1
-
-
Clostridium hylemonae DSM 15053
B4YSU1
-
-
Clostridium scindens
B0NI18
-
-
Clostridium scindens
P19412
-
-
Clostridium scindens ATCC 35704
B0NI18
-
-
Clostridium scindens VPI 12708
P19412
-
-
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
7alpha-hydroxy-3-oxochol-4-en-24-oate
-
735231
Clostridium hiranonis
3-oxochola-4,6-dien-24-oate
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oate
-
735231
Clostridium hylemonae
3-oxochola-4,6-dien-24-oate
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oate
-
735231
Clostridium scindens
3-oxochola-4,6-dien-24-oate
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oate
-
735231
Clostridium hylemonae DSM 15053
3-oxochola-4,6-dien-24-oate
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oate
-
735231
Clostridium scindens ATCC 35704
3-oxochola-4,6-dien-24-oate
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oate
-
735231
Clostridium scindens VPI 12708
3-oxochola-4,6-dien-24-oate
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oate
-
735231
Clostridium hiranonis DSM 13275
3-oxochola-4,6-dien-24-oate
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
-
735231
Clostridium hiranonis
3-oxochola-4,6-dien-24-oyl-CoA
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
-
735231
Clostridium hylemonae
3-oxochola-4,6-dien-24-oyl-CoA
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
-
735231
Clostridium scindens
3-oxochola-4,6-dien-24-oyl-CoA
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
-
735231
Clostridium hylemonae DSM 15053
3-oxochola-4,6-dien-24-oyl-CoA
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
-
735231
Clostridium scindens ATCC 35704
3-oxochola-4,6-dien-24-oyl-CoA
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
-
735231
Clostridium scindens VPI 12708
3-oxochola-4,6-dien-24-oyl-CoA
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
-
735231
Clostridium hiranonis DSM 13275
3-oxochola-4,6-dien-24-oyl-CoA
-
-
-
?
Turnover Number [1/s]
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
4.4
-
7alpha-hydroxy-3-oxochol-4-en-24-oate
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium hiranonis
12.6
-
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium hylemonae
15
-
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium scindens
18
-
7alpha-hydroxy-3-oxochol-4-en-24-oate
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium scindens
18.6
-
7alpha-hydroxy-3-oxochol-4-en-24-oate
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium hylemonae
22.2
-
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium hiranonis
24
-
7alpha-hydroxy-3-oxochol-4-en-24-oate
25°C in 20 mM Tris-HCl pH 7.3
Clostridium scindens
98
-
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
25°C in 20 mM Tris-HCl pH 7.3
Clostridium scindens
Crystallization (Commentary) (protein specific)
Crystallization
Organism
-
Clostridium hiranonis
-
Clostridium hylemonae
-
Clostridium scindens
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
D106N
inactive mutant
Clostridium scindens
D35N
inactive mutant
Clostridium scindens
H83N
inactive mutant
Clostridium scindens
R146Q
inactive mutant
Clostridium scindens
Y126F
strongly reduced activity compared to wild type
Clostridium scindens
Y30F
inactive mutant
Clostridium scindens
Y54A
inactive mutant
Clostridium scindens
Y54F
reduced activity compared to wild type
Clostridium scindens
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.2
-
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium scindens
4
-
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
25°C in 20 mM Tris-HCl pH 7.3
Clostridium scindens
9.9
-
7alpha-hydroxy-3-oxochol-4-en-24-oate
25°C in 20 mM Tris-HCl pH 7.3
Clostridium scindens
11
-
7alpha-hydroxy-3-oxochol-4-en-24-oate
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium scindens
11.4
-
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium hylemonae
21
-
7alpha-hydroxy-3-oxochol-4-en-24-oate
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium hylemonae
25
-
7alpha-hydroxy-3-oxochol-4-en-24-oate
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium hiranonis
208
-
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium hiranonis
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
Clostridium hiranonis
-
3-oxochola-4,6-dien-24-oyl-CoA
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
Clostridium hylemonae
-
3-oxochola-4,6-dien-24-oyl-CoA
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
Clostridium scindens
-
3-oxochola-4,6-dien-24-oyl-CoA
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
Clostridium hylemonae DSM 15053
-
3-oxochola-4,6-dien-24-oyl-CoA
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
Clostridium scindens ATCC 35704
-
3-oxochola-4,6-dien-24-oyl-CoA
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
Clostridium scindens VPI 12708
-
3-oxochola-4,6-dien-24-oyl-CoA
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
Clostridium hiranonis DSM 13275
-
3-oxochola-4,6-dien-24-oyl-CoA
-
-
?
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
7alpha-hydroxy-3-oxochol-4-en-24-oate
-
735231
Clostridium hiranonis
3-oxochola-4,6-dien-24-oate
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oate
-
735231
Clostridium hylemonae
3-oxochola-4,6-dien-24-oate
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oate
-
735231
Clostridium scindens
3-oxochola-4,6-dien-24-oate
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oate
-
735231
Clostridium hylemonae DSM 15053
3-oxochola-4,6-dien-24-oate
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oate
-
735231
Clostridium scindens ATCC 35704
3-oxochola-4,6-dien-24-oate
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oate
-
735231
Clostridium scindens VPI 12708
3-oxochola-4,6-dien-24-oate
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oate
-
735231
Clostridium hiranonis DSM 13275
3-oxochola-4,6-dien-24-oate
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
-
735231
Clostridium hiranonis
3-oxochola-4,6-dien-24-oyl-CoA
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
-
735231
Clostridium hylemonae
3-oxochola-4,6-dien-24-oyl-CoA
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
-
735231
Clostridium scindens
3-oxochola-4,6-dien-24-oyl-CoA
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
-
735231
Clostridium hylemonae DSM 15053
3-oxochola-4,6-dien-24-oyl-CoA
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
-
735231
Clostridium scindens ATCC 35704
3-oxochola-4,6-dien-24-oyl-CoA
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
-
735231
Clostridium scindens VPI 12708
3-oxochola-4,6-dien-24-oyl-CoA
-
-
-
?
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
-
735231
Clostridium hiranonis DSM 13275
3-oxochola-4,6-dien-24-oyl-CoA
-
-
-
?
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
4.4
-
7alpha-hydroxy-3-oxochol-4-en-24-oate
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium hiranonis
12.6
-
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium hylemonae
15
-
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium scindens
18
-
7alpha-hydroxy-3-oxochol-4-en-24-oate
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium scindens
18.6
-
7alpha-hydroxy-3-oxochol-4-en-24-oate
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium hylemonae
22.2
-
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium hiranonis
24
-
7alpha-hydroxy-3-oxochol-4-en-24-oate
25°C in 20 mM Tris-HCl pH 7.3
Clostridium scindens
98
-
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
25°C in 20 mM Tris-HCl pH 7.3
Clostridium scindens
KCat/KM [mM/s]
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
0.1
-
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium hiranonis
0.2
-
7alpha-hydroxy-3-oxochol-4-en-24-oate
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium hiranonis
0.9
-
7alpha-hydroxy-3-oxochol-4-en-24-oate
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium hylemonae
1.1
-
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium hylemonae
1.6
-
7alpha-hydroxy-3-oxochol-4-en-24-oate
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium scindens
2.4
-
7alpha-hydroxy-3-oxochol-4-en-24-oate
25°C in 20 mM Tris-HCl pH 7.3
Clostridium scindens
12.5
-
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium scindens
24.5
-
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
25°C in 20 mM Tris-HCl pH 7.3
Clostridium scindens
KCat/KM [mM/s] (protein specific)
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
0.1
-
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium hiranonis
0.2
-
7alpha-hydroxy-3-oxochol-4-en-24-oate
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium hiranonis
0.9
-
7alpha-hydroxy-3-oxochol-4-en-24-oate
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium hylemonae
1.1
-
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium hylemonae
1.6
-
7alpha-hydroxy-3-oxochol-4-en-24-oate
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium scindens
2.4
-
7alpha-hydroxy-3-oxochol-4-en-24-oate
25°C in 20 mM Tris-HCl pH 7.3
Clostridium scindens
12.5
-
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
25°C in 20 mM Tris-HCl, pH 7.3
Clostridium scindens
24.5
-
7alpha-hydroxy-3-oxochol-4-en-24-oyl-CoA
25°C in 20 mM Tris-HCl pH 7.3
Clostridium scindens
Other publictions for EC 4.2.1.106
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)
735231
Bhowmik
Structure and functional chara ...
Clostridium hiranonis, Clostridium hiranonis DSM 13275, Clostridium hylemonae, Clostridium hylemonae DSM 15053, Clostridium scindens, Clostridium scindens ATCC 35704, Clostridium scindens VPI 12708
Proteins
84
316-331
2016
-
-
-
3
8
-
-
8
-
-
-
8
-
8
-
-
-
-
-
-
-
-
16
-
-
-
-
8
-
-
-
-
-
-
-
-
-
-
-
4
8
-
-
-
-
8
-
-
-
8
-
-
-
-
-
-
-
-
16
-
-
-
-
8
-
-
-
-
-
-
-
-
8
8
658940
Kitahara
Clostridium hiranonis sp. nov. ...
Clostridium hiranonis
Int. J. Syst. Evol. Microbiol.
51
39-44
2001
-
-
-
-
-
-
-
-
-
-
-
1
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
657577
Wells
Identification and characteriz ...
Clostridium sp., Clostridium sp. TO-931
Appl. Environ. Microbiol.
66
1107-1113
2000
-
-
1
-
-
-
-
-
-
-
-
2
-
2
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
659658
Dawson
Expression and characterizatio ...
Eubacterium sp., Eubacterium sp. VPI 12708
J. Lipid Res.
37
1258-1267
1996
-
-
1
-
-
-
-
1
-
-
3
2
-
12
-
-
1
-
-
-
-
-
4
1
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
1
-
-
3
2
-
-
-
1
-
-
-
-
4
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
659119
Coleman
Biosynthesis of a novel bile a ...
Eubacterium sp., Eubacterium sp. VPI 12708
J. Biol. Chem.
262
4701-4707
1987
-
-
-
-
-
-
-
-
-
-
1
2
-
12
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
2
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-