BRENDA - Enzyme Database show
show all sequences of 2.3.1.165

Hidden function of catalytic domain in 6-methylsalicylic acid synthase for product release

Moriguchi, T.; Kezuka, Y.; Nonaka, T.; Ebizuka, Y.; Fujii, I.; J. Biol. Chem. 285, 15637-15643 (2010)

Data extracted from this reference:

Cloned(Commentary)
Commentary
Organism
functional expression of ATX in Escherichia coli strain BL21(DE3)-CodonPlus RIPL, expression of N-terminally His6-tagged ATX and its thioester hydrolase domain, and the truncated mutant enzyme form, THID
Aspergillus terreus
Engineering
Amino acid exchange
Commentary
Organism
H972A
site-directed mutagenesis, the reaction intermediate is bound to the thioester hydrolase domain of ATX formed in the presence of NADPH, and is released as 6-methylsalicylic acid by both the intact ATX and by truncated THID mutant in trans
Aspergillus terreus
additional information
construction of the THID mutant protein, a His-tagged 61 kDA 541-amino acid region containing thioester hydrolase domain and its downstream, THID shows catalytic activity to hydrolyze a model substrate 6-methylsalicylic acid-N-acetylcysteamine. The complementation of thioester domain-deficient mutant THm by THID protein in the recombinant Escherichia coli expression system
Aspergillus terreus
Inhibitors
Inhibitors
Commentary
Organism
Structure
additional information
no inhibition by phenylmethylsulfonyl fluoride, a serine protease-type thioesterase inhibitor at 1 mM
Aspergillus terreus
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
5.76
-
6-Methylsalicylate
release from enzyme/acyl-carrier-protein, recombinant His6-tagged THID mutant, pH 7.5, 25°C
Aspergillus terreus
12.3
-
6-Methylsalicylate
release from enzyme/acyl-carrier-protein, recombinant His6-tagged ATX, pH 7.5, 25°C
Aspergillus terreus
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
acetyl-CoA + 3 malonyl-CoA + NADPH + H+
Aspergillus terreus
-
6-methylsalicylate + 4 CoA + 3 CO2 + NADP+ + H2O
-
-
?
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Aspergillus terreus
-
gene atx
-
Purification (Commentary)
Commentary
Organism
recombinant N-terminally His6-tagged ATX and its thioester hydrolase domain, and the truncated mutant enzyme form, THID, from Escherichia coli strain BL21(DE3)-CodonPlus RIPL, by nickel affinity chromatography, and THID further by anion exchange chromatography, followed by gel filtration
Aspergillus terreus
Reaction
Reaction
Commentary
Organism
acetyl-CoA + 3 malonyl-CoA + NADPH + H+ = 6-methylsalicylate + 4 CoA + 3 CO2 + NADP+ + H2O
6-methylsalicylate synthesis proceeds without catalytic dehydration of the beta-hydroxy triketide intermediate by DH domain which is therefore not required for tetraketide formation. The subsequent aldol cyclization and aromatization of the tetraketide intermediate then form 6-methylsalicylate covalently bound to the ACP phosphopantetheine arm as the thioester. 6-methylsalicylate is released hydrolytically from ATX by the action of the domain hitherto called DH domain, i.e. the thioester hydrolase catalytic domain
Aspergillus terreus
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
acetyl-CoA + 3 malonyl-CoA + NADPH + H+
-
719864
Aspergillus terreus
6-methylsalicylate + 4 CoA + 3 CO2 + NADP+ + H2O
-
-
-
?
acetyl-CoA + 3 malonyl-CoA + NADPH + H+
ATX by itself can synthesize tetraketide and release 6-methylsalicylate as the free acid without involvement of serine protease-type thioesterase, the product 6-methylsalicylate is retained on the acyl-carrier-protein until its release from ATX, product-releasing mechanism, overview
719864
Aspergillus terreus
6-methylsalicylate + 4 CoA + 3 CO2 + NADP+ + H2O
-
-
-
?
additional information
the truncated enzyme, THID mutant, hydrolyzes the model substrate 6-methylsalicylic acid-N-acetylcysteamine
719864
Aspergillus terreus
?
-
-
-
-
Subunits
Subunits
Commentary
Organism
More
ATX does not have a conserved thioesterase GXSXG motif
Aspergillus terreus
tetramer
-
Aspergillus terreus
Temperature Optimum [°C]
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
25
-
assay at
Aspergillus terreus
Turnover Number [1/s]
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.0125
-
6-Methylsalicylate
release from enzyme/acyl-carrier-protein, recombinant His6-tagged THID mutant, pH 7.5, 25°C
Aspergillus terreus
0.023
-
6-Methylsalicylate
release from enzyme/acyl-carrier-protein, recombinant His6-tagged ATX, pH 7.5, 25°C
Aspergillus terreus
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7.5
-
assay at
Aspergillus terreus
Cofactor
Cofactor
Commentary
Organism
Structure
NADPH
-
Aspergillus terreus
Cloned(Commentary) (protein specific)
Commentary
Organism
functional expression of ATX in Escherichia coli strain BL21(DE3)-CodonPlus RIPL, expression of N-terminally His6-tagged ATX and its thioester hydrolase domain, and the truncated mutant enzyme form, THID
Aspergillus terreus
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
NADPH
-
Aspergillus terreus
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
H972A
site-directed mutagenesis, the reaction intermediate is bound to the thioester hydrolase domain of ATX formed in the presence of NADPH, and is released as 6-methylsalicylic acid by both the intact ATX and by truncated THID mutant in trans
Aspergillus terreus
additional information
construction of the THID mutant protein, a His-tagged 61 kDA 541-amino acid region containing thioester hydrolase domain and its downstream, THID shows catalytic activity to hydrolyze a model substrate 6-methylsalicylic acid-N-acetylcysteamine. The complementation of thioester domain-deficient mutant THm by THID protein in the recombinant Escherichia coli expression system
Aspergillus terreus
Inhibitors (protein specific)
Inhibitors
Commentary
Organism
Structure
additional information
no inhibition by phenylmethylsulfonyl fluoride, a serine protease-type thioesterase inhibitor at 1 mM
Aspergillus terreus
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
5.76
-
6-Methylsalicylate
release from enzyme/acyl-carrier-protein, recombinant His6-tagged THID mutant, pH 7.5, 25°C
Aspergillus terreus
12.3
-
6-Methylsalicylate
release from enzyme/acyl-carrier-protein, recombinant His6-tagged ATX, pH 7.5, 25°C
Aspergillus terreus
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
acetyl-CoA + 3 malonyl-CoA + NADPH + H+
Aspergillus terreus
-
6-methylsalicylate + 4 CoA + 3 CO2 + NADP+ + H2O
-
-
?
Purification (Commentary) (protein specific)
Commentary
Organism
recombinant N-terminally His6-tagged ATX and its thioester hydrolase domain, and the truncated mutant enzyme form, THID, from Escherichia coli strain BL21(DE3)-CodonPlus RIPL, by nickel affinity chromatography, and THID further by anion exchange chromatography, followed by gel filtration
Aspergillus terreus
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
acetyl-CoA + 3 malonyl-CoA + NADPH + H+
-
719864
Aspergillus terreus
6-methylsalicylate + 4 CoA + 3 CO2 + NADP+ + H2O
-
-
-
?
acetyl-CoA + 3 malonyl-CoA + NADPH + H+
ATX by itself can synthesize tetraketide and release 6-methylsalicylate as the free acid without involvement of serine protease-type thioesterase, the product 6-methylsalicylate is retained on the acyl-carrier-protein until its release from ATX, product-releasing mechanism, overview
719864
Aspergillus terreus
6-methylsalicylate + 4 CoA + 3 CO2 + NADP+ + H2O
-
-
-
?
additional information
the truncated enzyme, THID mutant, hydrolyzes the model substrate 6-methylsalicylic acid-N-acetylcysteamine
719864
Aspergillus terreus
?
-
-
-
-
Subunits (protein specific)
Subunits
Commentary
Organism
More
ATX does not have a conserved thioesterase GXSXG motif
Aspergillus terreus
tetramer
-
Aspergillus terreus
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
25
-
assay at
Aspergillus terreus
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.0125
-
6-Methylsalicylate
release from enzyme/acyl-carrier-protein, recombinant His6-tagged THID mutant, pH 7.5, 25°C
Aspergillus terreus
0.023
-
6-Methylsalicylate
release from enzyme/acyl-carrier-protein, recombinant His6-tagged ATX, pH 7.5, 25°C
Aspergillus terreus
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7.5
-
assay at
Aspergillus terreus
General Information
General Information
Commentary
Organism
evolution
6-methylsalicylic acid synthase belongs to the iterative type I polyketide synthases
Aspergillus terreus
physiological function
6-methylsalicylic acid synthase from Aspergillus terreus is not involved in dehydration of the beta-hydroxytriketide intermediate tethered on the acyl carrier protein but catalyzes thioester hydrolysis to release the product from the acyl carrier protein, via its thioester hydrolase domain, a product-releasing domain that is located in the middle of a multidomain iterative type I polyketide synthase, ATX by itself can synthesize tetraketide and release 6-methylsalicylate as the free acid without involvement of serine protease-type thioesterase, product-releasing mechanism, overview
Aspergillus terreus
General Information (protein specific)
General Information
Commentary
Organism
evolution
6-methylsalicylic acid synthase belongs to the iterative type I polyketide synthases
Aspergillus terreus
physiological function
6-methylsalicylic acid synthase from Aspergillus terreus is not involved in dehydration of the beta-hydroxytriketide intermediate tethered on the acyl carrier protein but catalyzes thioester hydrolysis to release the product from the acyl carrier protein, via its thioester hydrolase domain, a product-releasing domain that is located in the middle of a multidomain iterative type I polyketide synthase, ATX by itself can synthesize tetraketide and release 6-methylsalicylate as the free acid without involvement of serine protease-type thioesterase, product-releasing mechanism, overview
Aspergillus terreus
Other publictions for EC 2.3.1.165
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)
735433
Parascandolo
Insights into 6-methylsalicyli ...
Penicillium griseofulvum
Angew. Chem. Int. Ed. Engl.
55
3463-3467
2016
-
-
1
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
735950
Petersen
Investigation of a 6-MSA synth ...
Aspergillus aculeatus
ChemBioChem
16
2200-2204
2015
-
-
-
-
-
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
735929
Holm
Molecular and chemical charact ...
Aspergillus niger, Aspergillus niger ATCC 1015
Chem. Biol.
21
519-529
2014
-
-
1
-
-
-
-
-
-
-
-
-
-
4
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
719864
Moriguchi
Hidden function of catalytic d ...
Aspergillus terreus
J. Biol. Chem.
285
15637-15643
2010
-
-
1
-
2
-
1
2
-
-
-
1
-
2
-
-
1
1
-
-
-
-
3
2
1
-
-
2
1
-
-
1
-
-
-
-
-
1
1
-
2
-
-
1
-
2
-
-
-
1
-
-
-
1
-
-
-
-
3
2
1
-
-
2
1
-
-
-
-
2
2
-
-
-
690561
Panagiotou
Studies on the production of f ...
Penicillium griseofulvum
Appl. Environ. Microbiol.
75
2212-2220
2009
-
2
1
-
-
-
-
-
-
-
-
-
-
6
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
703577
Sanzani
Effect of quercetin and umbell ...
Penicillium expansum
Eur. J. Plant Pathol.
125
223-233
2009
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
1
1
2
-
-
703824
Bacha
Cloning and characterization o ...
Aspergillus westerdijkiae
Fungal Genet. Biol.
46
742-749
2009
-
-
-
-
1
-
-
-
-
-
-
1
-
5
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
703206
Moriguchi
Domain-domain interactions in ...
Aspergillus terreus, Penicillium griseofulvum
ChemBioChem
9
1207-1212
2008
-
-
1
-
5
-
-
-
-
-
1
-
-
3
-
-
-
-
-
-
-
-
2
2
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
5
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
2
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
705512
Wattanachaisaereekul
Production of the polyketide 6 ...
Penicillium griseofulvum
Metab. Eng.
10
246-254
2008
-
2
1
-
1
-
-
-
-
-
-
-
-
4
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
2
1
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
687164
Puel
The inability of Byssochlamys ...
Byssochlamys nivea, Byssochlamys nivea NRRL 2615
Int. J. Food Microbiol.
115
131-139
2007
-
1
1
-
-
-
-
-
-
-
-
-
-
7
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
671762
Shao
Cloning and characterization o ...
Streptomyces antibioticus
Biochem. Biophys. Res. Commun.
345
133-139
2006
-
-
1
-
-
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
672715
Wattanachaisaereekul
Optimization of heterologous p ...
Penicillium griseofulvum
Biotechnol. Bioeng.
97
893-900
2006
1
1
1
-
-
-
-
-
-
-
-
1
-
6
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
1
2
1
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
673079
Moriguchi
Analysis of subunit interactio ...
Aspergillus terreus
Chembiochem
7
1869-1874
2006
-
-
-
-
1
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
676009
Lu
A gene (pks2) encoding a putat ...
Glarea lozoyensis
Mol. Genet. Genomics
273
207-216
2005
-
-
1
-
-
-
-
-
-
-
-
-
-
4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
486452
Richardson
Tolerance and specificity of r ...
Penicillium griseofulvum
Metab. Eng.
1
180-187
1999
-
1
1
-
-
-
-
-
-
-
-
-
-
5
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
486449
Child
-
Inactivation of the polyketide ...
Penicillium griseofulvum
Biochem. J.
330
933-937
1998
-
-
-
-
-
-
2
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
486451
Campuzano
Incubation of 6-methylsalicyli ...
Penicillium griseofulvum
Biochem. Soc. Trans.
26
S284
1998
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
9
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
9
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
486450
Child
Structural similarities betwee ...
Penicillium griseofulvum
Biochemistry
35
12267-12274
1996
-
-
-
-
-
-
4
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
486447
Spencer
-
Purification and properties of ...
Penicillium griseofulvum, Penicillium griseofulvum NRRL 2159A
Biochem. J.
288
839-846
1992
-
-
-
-
-
1
1
4
-
-
2
-
-
4
-
-
1
1
-
-
1
1
6
1
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
-
-
1
-
1
-
4
-
-
2
-
-
-
-
1
-
-
1
1
6
1
-
-
-
-
1
1
-
-
-
-
-
-
-
-
486448
Spencer
Investigation of the mechanism ...
Penicillium griseofulvum
Biochemistry
31
9107-9116
1992
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
1
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
486446
Lam
In vitro stabilization of 6-me ...
Penicillium griseofulvum, Penicillium griseofulvum NRRL 2159A
Can. J. Microbiol.
34
30-37
1988
-
-
-
-
-
1
4
-
-
-
-
2
-
5
-
-
-
-
-
-
-
-
4
-
-
-
1
-
-
-
1
1
-
-
-
-
-
-
1
-
-
1
-
4
-
-
-
-
-
2
-
-
-
-
-
-
-
-
4
-
-
-
1
-
-
-
1
-
-
-
-
-
-
-
486445
Dimroth
6-Methylsalicylic acid synthet ...
Penicillium griseofulvum, Penicillium griseofulvum NRRL 2159A
Eur. J. Biochem.
68
591-596
1976
-
-
-
-
-
1
1
-
-
-
-
2
-
5
-
-
-
-
-
-
-
-
8
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
1
-
-
1
-
1
-
-
-
-
-
2
-
-
-
-
-
-
-
-
8
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-