BRENDA - Enzyme Database
show all sequences of 1.14.20.5

Functional characterization of a Plagiochasma appendiculatum flavone synthase I showing flavanone 2-hydroxylase activity

Han, X.J.; Wu, Y.F.; Gao, S.; Yu, H.N.; Xu, R.X.; Lou, H.X.; Cheng, A.X.; FEBS Lett. 588, 2307-2314 (2014)

Data extracted from this reference:

Cloned(Commentary)
Cloned (Commentary)
Organism
expression in Escherichia coli
Plagiochasma appendiculatum
Engineering
Protein Variants
Commentary
Organism
A120M
when the purified recombinant mutant enzyme is incubated with naringenin as substrate, the ratio of 2-hydroxynaringenin to apigenin is reduced. The mutant enzyme is able to oxidize dihydrokaempferol to kaempferol with 162% of the catalytic activity relative to wild-type enzyme
Plagiochasma appendiculatum
F146I
when the purified recombinant mutant enzyme is incubated with naringenin as substrate, the ratio of 2-hydroxynaringenin to apigenin is reduced. The mutant enzyme is able to oxidize dihydrokaempferol to kaempferol with 86% of the catalytic activity relative to wild-type enzyme
Plagiochasma appendiculatum
F146I/A120M
the enzyme activity of the double mutant is reduced to a level of about 10% of the activity of the wild type enzyme and the only product generated is apigenin
Plagiochasma appendiculatum
L311F
when the purified recombinant mutant enzyme is incubated with naringenin as substrate, the ratio of 2-hydroxynaringenin to apigenin is reduced. The mutant enzyme is able to oxidize dihydrokaempferol to kaempferol with 355% of the catalytic activity relative to wild-type enzyme
Plagiochasma appendiculatum
L311F/A120M
the double mutant catalyzes the formation of 2-hydroxynaringenin and apigenin, although the ratio of these two compounds iss lower than that generated by the wild type enzyme
Plagiochasma appendiculatum
L311F/F146I
the double mutant catalyzes the formation of 2-hydroxynaringenin and apigenin, although the ratio of these two compounds iss lower than that generated by the wild type enzyme
Plagiochasma appendiculatum
L311F/F146I/A120M
the activity of the triple mutant falls to about 5% of the wild type enzyme
Plagiochasma appendiculatum
L311F/F146I/Y240P
inactive mutant
Plagiochasma appendiculatum
L311F/F146I/Y240P/A120M
inactive mutant
Plagiochasma appendiculatum
L311F/Y240P
the double mutant exhibits less flavone synthase activity than either the wild type or the Y240P single mutant enzymes
Plagiochasma appendiculatum
Y240P
the mutant enzyme converts naringenin to apigenin without producing any 2-hydroxynaringenin. The mutant has a higher affinity (lower Km) with naringenin than the wild type enzyme, but the catalytic efficiency of the mutant is lower than that of wild type enzyme. The mutant enzyme is able to oxidize dihydrokaempferol to kaempferol with 104% of the catalytic activity relative to wild-type enzyme
Plagiochasma appendiculatum
Y240P/A120M
the enzyme activity of the double mutant is reduced to a level of about 10% of the activity of the wild type enzyme and the only product generated is apigenin. The mutant enzyme is able to oxidize dihydrokaempferol to kaempferol with 175% of the catalytic activity relative to wild-type enzyme
Plagiochasma appendiculatum
Y240P/F146I
the enzyme activity of the double mutant is reduced to a level of about 10% of the activity of the wild type enzyme and the only product generated is apigenin
Plagiochasma appendiculatum
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.0065
-
naringenin
pH 7.0, 35°C, mutant enzyme Y240P
Plagiochasma appendiculatum
0.0199
-
naringenin
pH 7.0, 35°C, wild-type enzyme
Plagiochasma appendiculatum
Organism
Organism
UniProt
Commentary
Textmining
Plagiochasma appendiculatum
A0A076U8J8
-
-
Purification (Commentary)
Purification (Commentary)
Organism
-
Plagiochasma appendiculatum
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
Substrate Product ID
dihydrokaempferol + 2-oxoglutarate + O2
the mutated enzymes L311F, Y240P, F146I, A120M and L311F–A120M are all able to oxidize dihydrokaempferol to kaempferol, with respective catalytic activities relative to the wild type level of 355%, 104%, 86%, 162% and 175%. L311F substitution increases the flavonol synthase activity
742512
Plagiochasma appendiculatum
kaempferol + succinate + CO2 + H2O
-
-
-
?
additional information
no activity with eriodictyol or dihydroquercetin
742512
Plagiochasma appendiculatum
?
-
-
-
-
naringenin + 2-oxoglutarate + O2
the enzyme catalyzes the conversion of naringenin to apigenin and 2-hydroxynaringenin. It is demonstrated that the enzyme is unable to catalyze the conversion of 2-hydroxynaringenin to apigenin. It is also confirmed that 2-hydroxynaringenin cannot be an intermediate product in the conversion of naringenin to apigenin, leaving the mechanism of conversion unknown
742512
Plagiochasma appendiculatum
apigenin + 2-hydroxynaringenin + succinate + CO2 + H2O
-
-
-
?
Subunits
Subunits
Commentary
Organism
?
x * 61000, SDS-PAGE
Plagiochasma appendiculatum
Synonyms
Synonyms
Commentary
Organism
flavone synthase I
-
Plagiochasma appendiculatum
FNS I
-
Plagiochasma appendiculatum
Temperature Optimum [°C]
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
35
-
-
Plagiochasma appendiculatum
Turnover Number [1/s]
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.002
-
naringenin
pH 7.0, 35°C, mutant enzyme Y240P
Plagiochasma appendiculatum
0.027
-
naringenin
pH 7.0, 35°C, wild-type enzyme
Plagiochasma appendiculatum
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7
-
-
Plagiochasma appendiculatum
pI Value
Organism
Commentary
pI Value Maximum
pI Value
Plagiochasma appendiculatum
calculated from sequence
-
6.42
Cloned(Commentary) (protein specific)
Commentary
Organism
expression in Escherichia coli
Plagiochasma appendiculatum
Engineering (protein specific)
Protein Variants
Commentary
Organism
A120M
when the purified recombinant mutant enzyme is incubated with naringenin as substrate, the ratio of 2-hydroxynaringenin to apigenin is reduced. The mutant enzyme is able to oxidize dihydrokaempferol to kaempferol with 162% of the catalytic activity relative to wild-type enzyme
Plagiochasma appendiculatum
F146I
when the purified recombinant mutant enzyme is incubated with naringenin as substrate, the ratio of 2-hydroxynaringenin to apigenin is reduced. The mutant enzyme is able to oxidize dihydrokaempferol to kaempferol with 86% of the catalytic activity relative to wild-type enzyme
Plagiochasma appendiculatum
F146I/A120M
the enzyme activity of the double mutant is reduced to a level of about 10% of the activity of the wild type enzyme and the only product generated is apigenin
Plagiochasma appendiculatum
L311F
when the purified recombinant mutant enzyme is incubated with naringenin as substrate, the ratio of 2-hydroxynaringenin to apigenin is reduced. The mutant enzyme is able to oxidize dihydrokaempferol to kaempferol with 355% of the catalytic activity relative to wild-type enzyme
Plagiochasma appendiculatum
L311F/A120M
the double mutant catalyzes the formation of 2-hydroxynaringenin and apigenin, although the ratio of these two compounds iss lower than that generated by the wild type enzyme
Plagiochasma appendiculatum
L311F/F146I
the double mutant catalyzes the formation of 2-hydroxynaringenin and apigenin, although the ratio of these two compounds iss lower than that generated by the wild type enzyme
Plagiochasma appendiculatum
L311F/F146I/A120M
the activity of the triple mutant falls to about 5% of the wild type enzyme
Plagiochasma appendiculatum
L311F/F146I/Y240P
inactive mutant
Plagiochasma appendiculatum
L311F/F146I/Y240P/A120M
inactive mutant
Plagiochasma appendiculatum
L311F/Y240P
the double mutant exhibits less flavone synthase activity than either the wild type or the Y240P single mutant enzymes
Plagiochasma appendiculatum
Y240P
the mutant enzyme converts naringenin to apigenin without producing any 2-hydroxynaringenin. The mutant has a higher affinity (lower Km) with naringenin than the wild type enzyme, but the catalytic efficiency of the mutant is lower than that of wild type enzyme. The mutant enzyme is able to oxidize dihydrokaempferol to kaempferol with 104% of the catalytic activity relative to wild-type enzyme
Plagiochasma appendiculatum
Y240P/A120M
the enzyme activity of the double mutant is reduced to a level of about 10% of the activity of the wild type enzyme and the only product generated is apigenin. The mutant enzyme is able to oxidize dihydrokaempferol to kaempferol with 175% of the catalytic activity relative to wild-type enzyme
Plagiochasma appendiculatum
Y240P/F146I
the enzyme activity of the double mutant is reduced to a level of about 10% of the activity of the wild type enzyme and the only product generated is apigenin
Plagiochasma appendiculatum
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.0065
-
naringenin
pH 7.0, 35°C, mutant enzyme Y240P
Plagiochasma appendiculatum
0.0199
-
naringenin
pH 7.0, 35°C, wild-type enzyme
Plagiochasma appendiculatum
Purification (Commentary) (protein specific)
Commentary
Organism
-
Plagiochasma appendiculatum
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ID
dihydrokaempferol + 2-oxoglutarate + O2
the mutated enzymes L311F, Y240P, F146I, A120M and L311F–A120M are all able to oxidize dihydrokaempferol to kaempferol, with respective catalytic activities relative to the wild type level of 355%, 104%, 86%, 162% and 175%. L311F substitution increases the flavonol synthase activity
742512
Plagiochasma appendiculatum
kaempferol + succinate + CO2 + H2O
-
-
-
?
additional information
no activity with eriodictyol or dihydroquercetin
742512
Plagiochasma appendiculatum
?
-
-
-
-
naringenin + 2-oxoglutarate + O2
the enzyme catalyzes the conversion of naringenin to apigenin and 2-hydroxynaringenin. It is demonstrated that the enzyme is unable to catalyze the conversion of 2-hydroxynaringenin to apigenin. It is also confirmed that 2-hydroxynaringenin cannot be an intermediate product in the conversion of naringenin to apigenin, leaving the mechanism of conversion unknown
742512
Plagiochasma appendiculatum
apigenin + 2-hydroxynaringenin + succinate + CO2 + H2O
-
-
-
?
Subunits (protein specific)
Subunits
Commentary
Organism
?
x * 61000, SDS-PAGE
Plagiochasma appendiculatum
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
35
-
-
Plagiochasma appendiculatum
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.002
-
naringenin
pH 7.0, 35°C, mutant enzyme Y240P
Plagiochasma appendiculatum
0.027
-
naringenin
pH 7.0, 35°C, wild-type enzyme
Plagiochasma appendiculatum
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7
-
-
Plagiochasma appendiculatum
pI Value (protein specific)
Organism
Commentary
pI Value Maximum
pI Value
Plagiochasma appendiculatum
calculated from sequence
-
6.42
General Information
General Information
Commentary
Organism
physiological function
involved in flavonoid pathway
Plagiochasma appendiculatum
General Information (protein specific)
General Information
Commentary
Organism
physiological function
involved in flavonoid pathway
Plagiochasma appendiculatum
KCat/KM [mM/s]
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
0.305
-
naringenin
pH 7.0, 35°C, mutant enzyme Y240P
Plagiochasma appendiculatum
1.372
-
naringenin
pH 7.0, 35°C, wild-type enzyme
Plagiochasma appendiculatum
KCat/KM [mM/s] (protein specific)
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
0.305
-
naringenin
pH 7.0, 35°C, mutant enzyme Y240P
Plagiochasma appendiculatum
1.372
-
naringenin
pH 7.0, 35°C, wild-type enzyme
Plagiochasma appendiculatum
Other publictions for EC 1.14.20.5
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)
742512
Han
Functional characterization o ...
Plagiochasma appendiculatum
FEBS Lett.
588
2307-2314
2014
-
-
1
-
13
-
-
2
-
-
-
-
-
4
-
-
1
-
-
-
-
-
3
1
2
1
-
-
2
1
-
-
-
-
1
-
-
-
1
-
-
13
-
-
-
-
2
-
-
-
-
-
-
-
1
-
-
-
-
3
1
1
-
-
2
1
-
-
1
-
1
1
-
2
2
726253
Deguchi
Endogenous post-transcriptiona ...
Dahlia pinnata
Planta
237
1325-1335
2013
-
-
1
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
1
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
713237
Fliegmann
Flavone synthase II (CYP93B16) ...
no activity in Glycine max
Phytochemistry
71
508-514
2010
-
-
-
-
-
-
-
-
-
-
-
-
-
4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
699499
Jeon
Biological synthesis of 7-O-me ...
Populus deltoides
J. Microbiol. Biotechnol.
19
491-494
2009
-
1
1
-
-
-
-
-
-
-
1
1
-
3
-
-
-
-
-
-
-
-
1
-
2
-
-
-
-
1
-
-
-
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
685694
Yun
Expression of parsley flavone ...
Petroselinum crispum
Biosci. Biotechnol. Biochem.
72
968-973
2008
-
-
1
-
1
-
-
-
-
-
-
-
-
5
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
685800
Lee
Characterization of flavone sy ...
Oryza sativa
BMB Rep.
41
68-71
2008
-
-
1
-
-
-
-
-
-
1
1
-
-
5
-
-
1
-
-
-
-
-
2
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
1
1
-
-
-
-
-
-
-
-
1
1
-
-
-
-
1
-
-
-
-
2
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
699792
Jeong
-
Analysis of flavonoids and cha ...
Oryza sativa
J. Plant Biol.
51
97-101
2008
1
-
1
-
-
-
-
1
-
1
2
1
-
1
-
-
1
-
-
1
-
-
1
-
3
-
-
-
-
-
-
-
-
-
-
-
1
-
1
-
-
-
-
-
-
-
1
-
1
2
1
-
-
-
1
-
1
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
679173
Katsuyama
Synthesis of unnatural flavono ...
Petroselinum crispum
Chem. Biol.
14
613-621
2007
-
1
1
-
1
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
1
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
689577
Gebhardt
Evolution of flavone synthase ...
Petroselinum crispum
Plant Physiol.
144
1442-1454
2007
-
-
1
-
1
-
-
-
-
-
-
-
-
4
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
689585
Zhang
Flavone synthases from Medicag ...
Medicago truncatula
Plant Physiol.
144
741-751
2007
-
1
1
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
4
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
4
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
671519
Leonard
Expression of a soluble flavon ...
Petroselinum crispum
Appl. Microbiol. Biotechnol.
70
85-91
2006
-
-
1
-
-
-
-
-
-
-
-
-
-
7
-
-
-
-
-
-
-
-
2
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
671524
Miyahisa
Combinatorial biosynthesis of ...
Petroselinum crispum
Appl. Microbiol. Biotechnol.
71
53-58
2006
-
-
1
-
-
-
-
-
-
-
-
-
-
6
-
-
-
-
-
-
-
-
2
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
671436
Leonard
Investigation of two distinct ...
Petroselinum crispum
Appl. Environ. Microbiol.
71
8241-8248
2005
-
-
1
-
-
-
-
-
-
-
-
-
-
4
-
-
-
-
-
1
-
-
3
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
676365
Martens
Flavones and flavone synthases ...
Petroselinum crispum
Phytochemistry
66
2399-2407
2005
-
-
-
-
-
-
-
-
-
1
-
-
-
4
-
-
-
-
-
-
-
-
1
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
656956
Schroeder
Flavonoid methylation: a novel ...
Petroselinum crispum
Phytochemistry
65
1085-1094
2004
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
6
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
6
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
655572
Martens
Divergent evolution of flavono ...
Petroselinum crispum
FEBS Lett.
544
93-98
2003
-
-
1
-
-
-
-
-
-
-
-
-
-
4
-
-
-
-
-
1
-
-
2
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
654335
Lukacin
Purification and antigenicity ...
Petroselinum crispum
Arch. Biochem. Biophys.
393
177-183
2001
-
-
-
-
-
-
-
-
-
-
-
-
-
6
-
-
1
-
-
1
1
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
1
1
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
656942
Martens
Cloning of parsley flavone syn ...
Petroselinum crispum
Phytochemistry
58
43-46
2001
-
-
1
-
-
-
-
-
-
-
-
-
-
3
-
-
-
-
-
1
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
439113
Britsch
Purification and characterizat ...
Petroselinum crispum
Arch. Biochem. Biophys.
282
152-160
1990
1
-
-
-
-
-
4
3
1
1
1
-
-
2
-
-
1
-
-
1
1
-
3
1
3
-
-
-
-
1
-
-
-
2
1
-
1
-
-
-
-
-
-
-
4
2
3
1
1
1
-
-
-
-
1
-
1
1
-
3
1
-
-
-
-
1
-
-
1
-
-
-
-
-
-