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Enzyme Nomenclature
Information on EC 1.4.3.19 - glycine oxidase
for references in articles please use BRENDA:EC1.4.3.19
Word Map on EC 1.4.3.19
- 1.4.3.19
- subtilis
-
d-amino
- sarcosine
- amine
- flavoproteins
- fad
- flavin
-
flavoenzyme
-
deamination
- d-proline
- glyphosate
- glyoxylate
-
fad-dependent
- d-alanine
-
tryptophylquinone
-
homotetrameric
-
fad-binding
- 5-enolpyruvylshikimate-3-phosphate
-
rosetta
-
site-saturation
-
thios
-
fad-containing
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Specify your search results
The enzyme appears in viruses and cellular organisms
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EC NUMBER 
COMMENTARY 
1.4.3.19
-
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RECOMMENDED NAME
GeneOntology No.
glycine oxidase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2-iminoacetate + H2O = glyoxylate + NH3
(1b)
-
-
-
glycine + O2 = 2-iminoacetate + H2O2
(1a)
-
-
-
glycine + H2O + O2 = glyoxylate + NH3 + H2O2
overall reaction
-
-
-
glycine + H2O + O2 = glyoxylate + NH3 + H2O2
ternary complex sequential mechanism
-
glycine + H2O + O2 = glyoxylate + NH3 + H2O2
reaction mechanism involving an FADH2-imino acid complex, overview
-
glycine + H2O + O2 = glyoxylate + NH3 + H2O2
reaction mechanism involving an FADH2-imino acid complex, overview
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
oxidation
redox reaction
-
-
-
-
reduction
-
-
-
-
oxidation
-
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
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SYSTEMATIC NAME
IUBMB Comments
glycine:oxygen oxidoreductase (deaminating)
A flavoenzyme containing non-covalently bound FAD. The enzyme from Bacillus subtilis is active with glycine, sarcosine, N-ethylglycine, D-alanine, D-alpha-aminobutyrate, D-proline, D-pipecolate and N-methyl-D-alanine. It differs from EC 1.4.3.3, D-amino-acid oxidase, due to its activity on sarcosine and D-pipecolate. The intermediate 2-iminoacetate is used directly by EC 2.8.1.10, thiazole synthase.
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CAS REGISTRY NUMBER
COMMENTARY
39307-16-9
-
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
D-2-aminobutyrate + H2O + O2
2-oxobutyrate + H2O2
-
as active as sarcosine
-
-
?
D-2-aminobutyrate + H2O + O2
? + NH3 + H2O2
-
2.2% of the activity with sarcosine
-
-
?
D-aminobutanoate + H2O + O2
? + H2O2
-
30% of the activity with sarcosine
-
-
?
D-Ile + H2O + O2
3-methyl-2-oxopentanoate + H2O2
-
about 30% of the activity with sarcosine
-
-
?
D-Leu + H2O + O2
4-methyl-2-oxopentanoate + H2O2
-
about 35% of the activity with sarcosine
-
-
?
D-Pro + H2O + O2
? + H2O2
-
about 120% of the activity with sarcosine
-
-
?
D-Val + H2O + O2
3-methyl-2-oxobutanoate + H2O2
-
about 35% of the activity with sarcosine
-
-
?
D-Val + H2O + O2
? + NH3 + H2O2
-
4.8% of the activity with sarcosine
-
-
?
glycine ethyl ester + H2O + O2
?
-
30.7% activity compared to glycine
-
-
?
N-ethylglycine + H2O + O2
?
-
0.3% activity compared to glycine
-
-
?
N-methyl-D-Ala + H2O + O2
? + H2O2
-
75% of the activity with sarcosine
-
-
?
N-methyl-D-Ala + H2O + O2
? + NH3 + H2O2
-
16.9% of the activity with sarcosine
-
-
-
N-methyl-D-Ala + H2O + O2
pyruvate + methylamine + H2O2
-
about 110% of the activity with sarcosine
-
-
?
cyclopropylglycine + H2O + O2
?
enzyme is required for the biosynthesis of the thiazole moiety of thiamine diphosphate
-
-
?
D-4-hydroxyphenylglycine + H2O + O2
?
-
0.6% activity compared to glycine
-
-
?
D-4-hydroxyphenylglycine + H2O + O2
?
-
0.6% activity compared to glycine
-
-
?
D-Ala + H2O + O2
pyruvate + NH3 + H2O2
-
7.4% of the activity with sarcosine
-
-
?
D-Ala + H2O + O2
pyruvate + NH3 + H2O2
no activity with D-Ala
-
-
?
D-Ala + H2O + O2
pyruvate + NH3 + H2O2
-
about 115% of the activity with sarcosine
-
-
?
D-alanine + H2O + O2
pyruvate + NH3 + H2O2
-
0.2% activity compared to glycine
-
-
?
D-alanine + H2O + O2
pyruvate + NH3 + H2O2
-
0.2% activity compared to glycine
-
-
?
D-methionine + H2O + O2
4-(methylsulfanyl)-2-oxobutanoic acid + NH3 + H2O2
-
25% of the activity with sarcosine
-
-
?
D-methionine + H2O + O2
4-(methylsulfanyl)-2-oxobutanoic acid + NH3 + H2O2
-
-
-
-
?
D-methionine + H2O + O2
4-(methylsulfanyl)-2-oxobutanoic acid + NH3 + H2O2
-
-
-
-
?
D-norvaline + H2O + O2
2-oxopentanoic acid + NH3 + H2O2
-
-
-
-
?
D-phenylalanine + H2O + O2
phenylpyruvate + NH3 + H2O2
-
-
-
-
?
D-pipecolate + H2O + O2
? + H2O2
-
about 85% of the activity with sarcosine
-
-
?
D-pipecolate + H2O + O2
? + H2O2
-
70% of the activity with sarcosine
-
-
?
D-Pro + H2O + O2
? + NH3 + H2O2
-
15.1% of the activity with sarcosine
-
-
?
D-Pro + H2O + O2
? + NH3 + H2O2
-
110% of the activity with sarcosine
-
-
?
D-proline + H2O + O2
?
-
0.3% activity compared to glycine
-
-
?
D-valine + H2O + O2
3-methyl-2-oxobutanoic acid + NH3 + H2O2
-
-
-
-
?
D-valine + H2O + O2
3-methyl-2-oxobutanoic acid + NH3 + H2O2
-
-
-
-
?
glycine + H2O + O2
glyoxylate + NH3 + H2O2
-
-
-
-
?
glycine + H2O + O2
glyoxylate + NH3 + H2O2
-
-
free glycine oxidase forms the anionic red semiquinone upon photoreduction.This species is thermodynamically stable, as indicated by the large separation of the two single-electron reduction potentials of DeltaE 290 mV. The first potential is pH-independent, while the second is dependent. The midpoint reduction potential exhibits a 23.4 mV/pH unit slope, which is consistent with an overall two-electrons/one-proton transfer in the reduction to yield anionic reduced flavin. In the presence of glycolate and at pH 7.5 the potential for the semiquinone-reduced enzyme couple is shifted positively by about 160 mV, this favors a two-electron transfer compared to the free enzyme. Binding of glycolate and sulfite is also affected by pH
-
?
glycine + H2O + O2
glyoxylate + NH3 + H2O2
-
77.4% of the activity with sarcosine
-
-
?
glycine + H2O + O2
glyoxylate + NH3 + H2O2
-
85% of the activity with sarcosine
-
-
?
glycine + H2O + O2
glyoxylate + NH3 + H2O2
-
100% activity
-
-
?
glycine + O2 + H2O
glyoxylate + H2O2 + NH3
-
using samples of [2-RS-3H2,2-14C]-, [2-R-3H,2-14C]-, and [2-S-3H,2-14C]glycine, HSi is removed in the overall process. Incubation of the enzyme with [2-RS-3H2,2-14C]glycine under anaerobic conditions, when only the reducing half of the reaction can occur, leads to the recovery of 98.5% of the original glycine, which has the same 3H:14C ratio as the starting substrate. Isotope effects, overview
-
-
?
glycine + O2 + H2O
glyoxylate + H2O2 + NH3
-
using samples of [2-RS-3H2,2-14C]-, [2-R-3H,2-14C]-, and [2-S-3H,2-14C]glycine, HSi is removed in the overall process. Incubation of the enzyme with [2-RS-3H2,2-14C]glycine under anaerobic conditions, when only the reducing half of the reaction can occur, leads to the recovery of 98.5% of the original glycine, which has the same 3H:14C ratio as the starting substrate. Isotope effects, overview
-
-
?
glycine-ethyl-ester + H2O + O2
? + H2O2
-
90% of the activity with sarcosine
-
-
?
N-ethylglycine + H2O + O2
glyoxylate + ethylamine + H2O2
-
about 65% of the activity with sarcosine
-
-
?
N-ethylglycine + H2O + O2
glyoxylate + ethylamine + H2O2
-
120% of the activity with sarcosine
-
-
?
N-ethylglycine + H2O + O2
glyoxylate + ethylamine + H2O2
-
-
-
-
?
N-ethylglycine + H2O + O2
glyoxylate + NH3 + H2O2
-
85.3% of the activity with sarcosine
-
-
?
sarcosine + H2O + O2
?
-
0.1% activity compared to glycine
-
-
?
sarcosine + H2O + O2
glyoxylate + methylamine + H2O2
-
-
-
-
?
sarcosine + H2O + O2
glyoxylate + methylamine + H2O2
-
-
-
-
?
additional information
?
-
-
glycine oxidase is converted to a two-electron reduced form upon anaerobic reduction with the individual substrates and its reductive half-reaction is reversible
-
-
-
additional information
?
-
-
a general catabolic role of the enzyme on primary or secondary amines is excluded, because the expression of glycine oxidase is not inducible by Gly, sarcosine, or D-Ala as carbon or nitrogen source
-
-
-
additional information
?
-
-
the enzyme is strictly stereospecific as it only catalyzes the oxidation of the D-isomer
-
-
-
additional information
?
-
-
no activity with butilamine, benzilamine, D-histidine, D-leucine, L-proline, D-tryptophan, and D-tyrosine
-
-
-
additional information
?
-
-
substrate preference for amines of small size, like glycine, sarcosine, N-ethyl-glycine, and glycine-ethylester
-
-
-
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
cyclopropylglycine + H2O + O2
?
O31616
enzyme is required for the biosynthesis of the thiazole moiety of thiamine diphosphate
-
-
?
glycine ethyl ester + H2O + O2
?
-
30.7% activity compared to glycine
-
-
?
N-ethylglycine + H2O + O2
?
-
0.3% activity compared to glycine
-
-
?
D-4-hydroxyphenylglycine + H2O + O2
?
-
0.6% activity compared to glycine
-
-
?
D-4-hydroxyphenylglycine + H2O + O2
?
-
0.6% activity compared to glycine
-
-
?
D-alanine + H2O + O2
pyruvate + NH3 + H2O2
-
0.2% activity compared to glycine
-
-
?
D-alanine + H2O + O2
pyruvate + NH3 + H2O2
-
0.2% activity compared to glycine
-
-
?
D-proline + H2O + O2
?
-
0.3% activity compared to glycine
-
-
?
glycine + H2O + O2
glyoxylate + NH3 + H2O2
-
100% activity
-
-
?
sarcosine + H2O + O2
?
-
0.1% activity compared to glycine
-
-
?
additional information
?
-
-
a general catabolic role of the enzyme on primary or secondary amines is excluded, because the expression of glycine oxidase is not inducible by Gly, sarcosine, or D-Ala as carbon or nitrogen source
-
-
-
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
FAD
-
tight binding of coenzyme to the protein moiety, addition of exogenous FAD or FMN to the assay mixture does not increase enzyme activity
FAD
-
the isolated apoprotein species is present in solution as a monomer which rapidly recovers its tertiary structure and converts into the tetrameric holoenzyme following incubation with free FAD
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
no enhancement of enzyme activity in the presence of metal cations
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Co2+
there is a sharp decrease in activity when 1 mM Co2+ is added to the reaction assay
diethylether
73% residual activity after 30 min incubation at 30% (v/v)
ethyl acetate
19% residual activity after 30 min incubation at 30% (v/v)
methanol
54% residual activity after 30 min incubation at 30% (v/v)
N,N-Dimethyl formamide
17% residual activity after 30 min incubation at 30% (v/v)
Zn2+
there is a sharp decrease in activity when 1 mM Zn2+ is added to the reaction assay
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
4 - 10
D-alanine
-
apparent value for mutant enzyme M261Y, at 25°C and pH 8.5
20.6
D-alanine
-
in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate, at pH 8.5 and 37°C
217
D-alanine
-
mutant enzyme I15V, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C
315
D-alanine
-
wild type enzyme, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C
2000
D-alanine
-
Km above 2000 mM, apparent value for mutant enzyme M261H, at 25°C and pH 8.5
221.8
D-pipecolate
-
in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate, at pH 8.5 and 37°C
290
D-pipecolate
-
mutant enzyme I15V, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C
2000
D-pipecolate
-
wild type enzyme, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C
7.9
D-proline
-
mutant enzyme I15V, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C
8.9
D-proline
-
wild type enzyme, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C
9.3
D-proline
-
in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate, at pH 8.5 and 37°C
26
D-proline
-
apparent value for mutant enzyme H244F, at 25°C and pH 8.5; apparent value for mutant enzyme H244N, at 25°C and pH 8.5
0.25
glycine
-
in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate, at pH 8.5 and 37°C
0.4
glycine
-
mutant enzyme I15V, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C
0.6
glycine
-
wild type enzyme, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C
0.52
glycine-ethyl-ester
-
in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate, at pH 8.5 and 37°C
0.6
glycine-ethyl-ester
-
mutant enzyme I15V, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C
1.8
glycine-ethyl-ester
-
wild type enzyme, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C
0.22
N-ethylglycine
-
in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate, at pH 8.5 and 37°C
0.5
N-ethylglycine
-
mutant enzyme I15V, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C
2.8
N-ethylglycine
-
wild type enzyme, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C
0.45
sarcosine
-
in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate, at pH 8.5 and 37°C
0.51
sarcosine
-
mutant enzyme I15V, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C
0.6
sarcosine
-
wild type enzyme, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C
11
sarcosine
-
apparent value for mutant enzyme M261H, at 25°C and pH 8.5; apparent value for mutant enzyme M261Y, at 25°C and pH 8.5
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.33
D-alanine
-
kcat above 0.33 1/s, apparent value for mutant enzyme M261H, at 25°C and pH 8.5
0.47
D-alanine
-
apparent value for mutant enzyme M261Y, at 25°C and pH 8.5
0.75
D-alanine
-
apparent value for mutant enzyme H244N, at 25°C and pH 8.5
1.08
D-alanine
-
apparent value for mutant enzyme H244Q, at 25°C and pH 8.5
0.38
D-proline
-
apparent value for mutant enzyme M261Y, at 25°C and pH 8.5
0.62
D-proline
-
apparent value for mutant enzyme M261H, at 25°C and pH 8.5
1.23
D-proline
-
apparent value for mutant enzyme H244Q, at 25°C and pH 8.5
1.35
D-proline
-
apparent value for mutant enzyme H244F, at 25°C and pH 8.5
1.05
glyphosate
-
mutant G51S/A54R/H244A, pH 8.5, 25°C; mutant G51S/A54R, pH 8.5, 25°C
0.47
sarcosine
-
apparent value for mutant enzyme M261Y, at 25°C and pH 8.5
0.73
sarcosine
-
apparent value for mutant enzyme M261H, at 25°C and pH 8.5
1.08
sarcosine
-
apparent value for mutant enzyme H244F, at 25°C and pH 8.5
1.12
sarcosine
-
apparent value for mutant enzyme H244Q, at 25°C and pH 8.5
1.33
sarcosine
-
apparent value for mutant enzyme H244N, at 25°C and pH 8.5
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
11.85
14
cell free extract, when glycine is used as a substrate, at pH 8.0 and 37°C
960
after 68.6fold purification, when glycine is used as a substrate, at pH 8.0 and 37°C
1000
after 68.6fold purification, when D-alanine is used as a substrate, at pH 8.0 and 37°C
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
8.5
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7 - 9
about 75% activity at pH 7.0, more than 50% activity is retained between pH 7.5 and 8.5, about 60% activity at pH 9.0. The enzyme activity decreases sharply below pH 7.0 and above pH 9.0. There is a negligible amount of activity at pH below 5.0 and above 10.0
8 - 11
-
pH 8.0: about 55% of maximal activity, pH 11.0: about 55% of maximal activity, reaction with sarcosine
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
additional information
-
increase in activity with no evidence for any plateau or decrease up to 60°C, reaction with sarcosine
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
15 - 60
-
increase in activity with no evidence for any plateau or decrease up to 60°C, reaction with sarcosine
40 - 55
the enzyme exhibits almost 80% of its activity at temperatures 40 and 55°C. The activity sharply decreases above 55°C
60
-
activation during the first 15 min, with slow decrease thereafter
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pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6
-
theoretical value, His-tagged chimeric enzyme; theoretical value, wild-type enzyme
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PDB
SCOP
CATH
UNIPROT
ORGANISM
Bacillus subtilis (strain 168);
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
40000
40762
-
x * 40763, recombinant enzyme, mass spectrometry, x * 40762, sequence calculation
40763
-
x * 40763, recombinant enzyme, mass spectrometry, x * 40762, sequence calculation
42000
43000
160000
172000
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
homotetramer
tetramer
additional information
-
the isolated apoprotein of glycine oxidase shows high protein fluorescence, high exposure of hydrophobic surfaces, and low temperature stability as compared to the holoenzyme
?
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x * 40763, recombinant enzyme, mass spectrometry, x * 40762, sequence calculation
?
-
x * 40763, recombinant enzyme, mass spectrometry, x * 40762, sequence calculation
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homotetramer
4 * 40000, SDS-PAGE; 4 * 40760, MALDI-TOF mass spectrometry; 4 * 40761, calculated from amino acid sequence
homotetramer
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4 * 40000, SDS-PAGE; 4 * 40760, MALDI-TOF mass spectrometry; 4 * 40761, calculated from amino acid sequence
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tetramer
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4 * 46600, recombinant glycine oxidase, SDS-PAGE; 4 * 49400, recombinant His-tagged chimeric glycine oxidase
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Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4 - 9
-
a great decrease in stability is only produced at pH values below pH 4.0, shows good stability between pH 6.0 and 9.0
689934
6.5 - 10
-
the tetrameric oligomeric state of the holoenzyme is not affected within this range
696494
6.5 - 9.5
6.5 - 9.5
-
the tetrameric state of the enzyme is not affected by pH in the pH range 6.5-9.5
656226
6.5 - 9.5
-
the incubation of both enzymes for 1 h shows highest stability between pH 6.5 and pH 9.5, the mutant enzyme is also stable at pH below pH 6.5 retaining about 70% of its initial activity, this stability at a lower pH is in contrast to the sharp decrease in pH-stability showed by wild type GOX which retains only about 37% of the initial activity
687878
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
34.7
-
melting temperature, mutant G51R/A54R, determined by following protein and flavin fluorescence
41.3
-
melting temperature, mutant G51R, determined by following protein and flavin fluorescence
44.8
-
melting temperature, mutant A54R, determined by following protein and flavin fluorescence
45
45.3
-
melting temperature, mutant G51S/A54R, determined by following protein and flavin fluorescence
45.7
-
melting temperature, mutant G51S/A54R/H244A, determined by following protein and flavin fluorescence
56.9
60 - 80
-
at 60°C, the I15V mutant shows a similar profile than the wild type enzyme but with lower residual activity after 20 min, at 65°C the mutant goes through activation (160%) during the first 15 min, the activity then falls with a time-dependent trend and reaches a half-life value similar to the wild type enzyme, at higher temperatures (70-80°C) the activity decreases rapidly in both cases, but with half-life value higher for I15V mutant than for wild type enzyme
95
-
heat treatment of the protein at 95°C for 5 min results in the release of the FAD cofactor
56.9
-
melting temperature, wild-type, determined by following protein and flavin fluorescence
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
tetrameric enzyme is strongly resistant to proteolysis, only the N-terminus is sensitive to proteases
-
thiocyanate, up to 1 mM, decreases stability of the tetrameric enzyme in solution
-
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80°C, 75 mM sodium diphosphate buffer pH 8.5 containing 10% glycerol, several months, no loss of activity
-
-80°C, 75 mM sodium diphosphate, pH 8.5, 10% glycerol, stable for months
-
-80°C, pH 7.5, both glycine oxidase and His-tagged glycine oxidase are stable for months
-
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
100 kDa ultrafiltration, HisTrap FF Ni2+-chelating affinity column chromatography, and phenyl-Sepharose FF hydrophobic column chromatography
-
ammonium sulfate precipitation, Resource Q column chromatography and Resource ISO column chromatography
HiTrap FF chelating affinity column chromatography and phenyl-Sepharose FF hydrophobic column chromatography
-
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning of the glycine oxidase gene from Bacillus subtilis ATCC 6633 into the Rosetta Escherichia coli strain which contains the pRARE plasmid, inserting of the gene into the pET28a expression vector
-
expressed in Escherichia coli Rosetta (DE3) cells
expressed in Escherichia coli Rosetta (DE3) cells Rosetta under the strong inducible T7 promotor of the pET28a vector
-
expression in Escherichia coli
production of a recombinant plasmid, pT7-GO by insertion of the DNA encoding for glycine oxidase into the multiple cloning site of the expression vector pT7.7. The pT7-glycine oxidase encodes a fully active fusion protein with six additional residues at the N-terminus of glycine oxidase. In BL21(DE3)pLysS Escherichia coli cells, and under optimal isopropyl thio-beta-D-galactoside induction conditions, soluble and active chimeric glycine oxidase is expressed up to 1.14 U per mg of cell. An N-terminally His-tagged glycine oxidase is also successfully expressed in Escherichia coli as a soluble protein and a fully active holoenzyme
-
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
A45H/G300C
-
monomeric in solution. Upon incubation of apoprotein with FAD, neither FAD binding nor enzymatic activity is observed. Slow elimination of urea from partially unfolded mutant in presence of a large excess of FAD and 30% glyccerol does not produce the holoenzyme
G300C
-
upon incubation of apoprotein with FAD, neither FAD binding nor enzymatic activity is observed. Slow elimination of urea from partially unfolded mutant in presence of a large excess of FAD and 30% glycerol does not produce the holoenzyme
G51R
-
4000fold increase in the specificity constant for substrate glyphosate
G51R/A54R
-
5800fold increase in the specificity constant for substrate glyphosate
G51S/A54R
-
3100fold increase in the specificity constant for substrate glyphosate
G51S/A54R/H244A
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210fold increase in catalytic activity and 15000fold increase in the specificity constant for substrate glyphosate. The alpha2-alpha3-loop assumes a different conformation, residue R54 may be the key residue in stabilizing glyphosate binding
H244F
-
shows increased kcat value for glycine compared to the wild type enzyme, the mutation does not affect the expression of glycine oxidase and the physicochemical properties of bound FAD
H244N
-
shows increased kcat value for glycine compared to the wild type enzyme, the mutation does not affect the expression of glycine oxidase and the physicochemical properties of bound FAD
H244Q
-
shows increased kcat value compared to the wild type enzyme, the mutation does not affect the expression of glycine oxidase and the physicochemical properties of bound FAD
I15V
-
the mutant exhibits a 7fold higher specific activity compared to the wild type enzyme
M261H
M261Y
Y241H
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100fold increase in the specificity constant for substrate glyphosate
M261H
-
shows decreased kcat value for glycine compared to the wild type enzyme, the mutation does not affect the expression of glycine oxidase and the physicochemical properties of bound FAD
M261Y
-
shows decreased kcat value for glycine compared to the wild type enzyme, the mutation does not affect the expression of glycine oxidase and the physicochemical properties of bound FAD
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Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
the isolated apoprotein species is present in solution as a monomer which rapidly recovers its tertiary structure and converts into the tetrameric holoenzyme following incubation with free FAD. The reconstitution process follows a particular two-stage process, the spectral properties of the reconstituted holoenzyme are virtually indistinguishable from those observed with native glycine oxidase, while the activity is only recovered to about 50%. The urea-induced unfolding process of glycine oxidase can be considered as a two-step process. Only a single transition at 4.5 M urea concentration is observed for the apoprotein form. The chemical denaturation of glycine oxidase holoenzyme is partially reversible
-
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LINKS TO OTHER DATABASES (specific for EC-Number 1.4.3.19)
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