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Information on EC 1.4.3.19 - glycine oxidase and Organism(s) Bacillus subtilis and UniProt Accession O31616
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1.4.3.19 glycine oxidaseIUBMB Comments
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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Word Map
- 1.4.3.19
-
d-amino
- sarcosine
- flavoproteins
- glyphosate
- flavin
- fad
-
tryptophylquinone
-
flavoenzyme
-
herbicide
- d-proline
-
homotetrameric
- d-alanine
-
pseudoalteromonas
- luteoviolacea
-
protein-derived
-
thios
-
fad-dependent
-
site-saturation
-
fad-binding
-
quinoprotein
- ttq
-
half-reaction
- 5-enolpyruvylshikimate-3-phosphate
-
fad-containing
The taxonomic range for the selected organisms is: Bacillus subtilis
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Reaction Schemes
hide(Overall reactions are displayed. Show all >>)
Synonyms
glycine oxidase, glyox, plgoxa, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
GO
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
redox reaction
-
-
-
-
oxidation
reduction
-
-
-
-
oxidation
-
-
-
-
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.
CAS REGISTRY NUMBER
COMMENTARY
39307-16-9
-
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-Ala + H2O + O2
? + NH3 + H2O2
40% of the activity with sarcosine
-
-
?
D-aminobutanoate + H2O + O2
? + H2O2
30% of the activity with sarcosine
-
-
?
D-methionine + H2O + O2
4-(methylsulfanyl)-2-oxobutanoic acid + NH3 + H2O2
25% of the activity with sarcosine
-
-
?
D-pipecolate + H2O + O2
? + H2O2
70% of the activity with sarcosine
-
-
?
D-Pro + H2O + O2
? + NH3 + H2O2
110% of the activity with sarcosine
-
-
?
glycine-ethyl-ester + H2O + O2
? + H2O2
90% of the activity with sarcosine
-
-
?
glyphosate + H2O + O2
? + H2O2
110% of the activity with sarcosine
-
-
?
glyphosate + H2O + O2
glyoxylate + aminomethylphosphonic acid + NH3 + H2O2
-
-
-
?
N-methyl-D-Ala + H2O + O2
? + H2O2
75% of the activity with sarcosine
-
-
?
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-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-pipecolate + H2O + O2
? + H2O2
-
about 85% 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
-
-
?
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
-
-
?
sarcosine + H2O + O2
glyoxylate + methylamine + H2O2
-
-
-
-
?
cyclopropylglycine + H2O + O2
?
enzyme is required for the biosynthesis of the thiazole moiety of thiamine diphosphate
-
-
?
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
85% of the activity with sarcosine
-
-
?
N-ethylglycine + H2O + O2
glyoxylate + ethylamine + H2O2
120% of the activity with sarcosine
-
-
?
D-Ala + H2O + O2
pyruvate + NH3 + H2O2
-
7.4% of the activity with sarcosine
-
-
?
D-Ala + H2O + O2
pyruvate + NH3 + H2O2
-
about 115% of the activity with sarcosine
-
-
?
D-Pro + H2O + O2
? + NH3 + H2O2
-
15.1% of the activity with sarcosine
-
-
?
glycine + H2O + O2
glyoxylate + NH3 + H2O2
-
-
-
-
?
glycine + H2O + O2
glyoxylate + NH3 + H2O2
-
77.4% of the activity with sarcosine
-
-
?
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
-
-
?
N-ethylglycine + H2O + O2
glyoxylate + ethylamine + H2O2
-
85.3% of the activity with sarcosine
-
-
?
N-ethylglycine + H2O + O2
glyoxylate + ethylamine + H2O2
-
about 65% of the activity with sarcosine
-
-
?
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
?
-
-
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
?
-
-
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
?
-
-
the enzyme is strictly stereospecific as it only catalyzes the oxidation of the D-isomer
-
-
?
NATURAL SUBSTRATE
NATURAL 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
cyclopropylglycine + H2O + O2
?
enzyme is required for the biosynthesis of the thiazole moiety of thiamine diphosphate
-
-
?
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
?
-
-
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
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
FAD
-
tight binding of coenzyme to the protein moiety, addition of exogenous FAD or FMN to the assay mixture does not increase enzyme activity
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
no enhancement of enzyme activity in the presence of metal cations
INHIBITOR
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)
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
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
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
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
0.6
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
0.5
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
0.51
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
4 - 10
D-alanine
-
apparent value for mutant enzyme M261Y, at 25°C and pH 8.5
2000
D-alanine
-
Km above 2000 mM, apparent value for mutant enzyme M261H, at 25°C and pH 8.5
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
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
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
1000
after 68.6fold purification, when D-alanine is used as a substrate, at pH 8.0 and 37°C
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
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
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
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
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
40762
-
x * 40763, recombinant enzyme, mass spectrometry, x * 40762, sequence calculation
40763
-
x * 40763, recombinant enzyme, mass spectrometry, x * 40762, sequence calculation
42000
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
homotetramer
?
-
x * 40763, recombinant enzyme, mass spectrometry, x * 40762, sequence calculation
homotetramer
tetramer
additional information
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
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
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
PROTEIN VARIANTS
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
A54E
the mutant shows reduced catalytic efficiency with glycine compared to the wild type enzyme
A54R
A54R/H244K
the mutant shows reduced catalytic efficiency with glycine compared to the wild type enzyme
A54R/H244K/M261R
the mutant shows reduced catalytic efficiency with glycine compared to the wild type enzyme
agriculture
expression of an evolved engineered variant of glycine oxidase leads to glyphosate resistance in alfalfa
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
G51H
the mutant shows reduced catalytic efficiency with glycine compared to the wild type enzyme
G51Q
the mutant shows reduced catalytic efficiency with glycine compared to the wild type enzyme
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
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
H244A
H244K
the mutant shows increased catalytic efficiency with glycine compared to the wild type enzyme
H244K/M261R
the variant shows a 5.4fold increase in maximal activity on glycine compared to the wild type enzyme
H244N
the mutant shows reduced catalytic efficiency with glycine compared to the wild type enzyme
H244Q
the mutant shows increased catalytic efficiency with glycine compared to the wild type enzyme
H244R
the mutant shows increased catalytic efficiency with glycine compared to the wild type enzyme
I15V
the mutant exhibits a 7fold higher specific activity compared to the wild type enzyme
L301V
improvement in residual activity after incubation of 60 min at 60°C to 12.9%
M261I
the mutant shows reduced catalytic efficiency with glycine compared to the wild type enzyme
M261R
the mutant shows reduced catalytic efficiency with glycine compared to the wild type enzyme
M49I
the mutant shows increased catalytic efficiency with glycine compared to the wild type enzyme
M49L
the mutant shows reduced catalytic efficiency with glycine compared to the wild type enzyme
M49L/A54R/H244K
the mutant shows reduced catalytic efficiency with glycine compared to the wild type enzyme
M49T
the mutant shows reduced catalytic efficiency with glycine compared to the wild type enzyme
T42A
improvement in residual activity after incubation of 60 min at 60°C to 18.7%
T42A/C245S/L301V
improvement in residual activity after incubation of 60 min at 60°C to 74.0%. The mutant enzyme retains most of its enzymatic activity during storage for over a year at 4°C
T42S
improvement in residual activity after incubation of 60 min at 60°C to 31.5%
T42S/C245S/L301V
improvement in residual activity after incubation of 60 min at 60°C to 55.7%
Y241H
100fold increase in the specificity constant for substrate glyphosate
Y246W
the mutant shows reduced catalytic efficiency with glycine compared to the wild type enzyme
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
M261H
M261Y
A54R
the mutant shows reduced catalytic efficiency with glycine compared to the wild type enzyme
H244A
the mutant shows reduced catalytic efficiency with glycine compared to the wild type enzyme
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
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
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
45.3
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
60 min, residual activity of wild-type is almost absent, activity of C245S mutant is 4.4%
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
45
56.9
melting temperature, wild-type, determined by following protein and flavin fluorescence
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
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, pH 7.5, both glycine oxidase and His-tagged glycine oxidase are stable for months
-
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
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
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 Rosetta under the strong inducible T7 promotor of the pET28a vector
-
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
-
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
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
diagnostics
a rapid and simple glycine analysis method is developed using a stable glycine oxidase mutant (T42 A/C245 S/L301V). Glycine analysis is important in research fields such as physiology and healthcare because the concentration of glycine in human plasma has been reported to change with various disorders
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Nishiya, Y.; Imanaka, T.
Purification and characterization of a novel glycine oxidase from Bacillus subtilis
FEBS Lett.
438
263-266
1998
Bacillus subtilis
Molla, G.; Motteran, L.; Job, V.; Pilone, M.S.; Pollegioni, L.
Kinetic mechanisms of glycine oxidase from Bacillus subtilis
Eur. J. Biochem.
270
1474-1482
2003
Bacillus subtilis
Settembre, E.C.; Dorrestein, P.C.; Park, J.H.; Augustine, A.M.; Begley, T.P.; Ealick, S.E.
Structural and mechanistic studies on ThiO, a glycine oxidase essential for thiamin biosynthesis in Bacillus subtilis
Biochemistry
42
2971-2981
2003
Bacillus subtilis (O31616), Bacillus subtilis
Job, V.; Molla, G.; Pilone, M.S.; Pollegioni, L.
Overexpression of a recombinant wild-type anf His-tagged Bacillus subtilis glycine oxidase in Escherichia coli
Eur. J. Biochem.
269
1456-1463
2002
Bacillus subtilis
Mrtl, M.; Diedrichs, K.; Welte, W.; Molla, G.; Motteran, L.; Andriolo, G.; Pilone, M.S.; Pollegioni, L.
Structure-function correlation in glycine oxidase from Bacillus subtilis
J. Biol. Chem.
279
29718-29727
2004
Bacillus subtilis (O31616), Bacillus subtilis
Martinez-Martinez, I.; Navarro-Fernandez, J.; Lozada-Ramirez, J.D.; Garcia-Carmona, F.; Sanchez-Ferrer, A.
Maximization of production of his-tagged glycine oxidase and its M261 mutant proteins
Biotechnol. Prog.
22
647-652
2006
Bacillus subtilis
Boselli, A.; Rosini, E.; Marcone, G.L.; Sacchi, S.; Motteran, L.; Pilone, M.S.; Pollegioni, L.; Molla, G.
Glycine oxidase from Bacillus subtilis: role of histidine 244 and methionine 261
Biochimie
89
1372-1380
2007
Bacillus subtilis
Martinez-Martinez, I.; Kaiser, C.; Rohde, A.; Ellert, A.; Garcia-Carmona, F.; Sanchez-Ferrer, A.; Luttmann, R.
High-level production of Bacillus subtilis glycine oxidase by fed-batch cultivation of recombinant Escherichia coli Rosetta (DE3)
Biotechnol. Prog.
23
645-651
2007
Bacillus subtilis
Martinez-Martinez, I.; Navarro-Fernandez, J.; Garcia-Carmona, F.; Sanchez-Ferrer, A.
Implication of a mutation in the flavin binding site on the specific activity and substrate specificity of glycine oxidase from Bacillus subtilis produced by directed evolution
J. Biotechnol.
133
1-8
2008
Bacillus subtilis (O31616), Bacillus subtilis
Caldinelli, L.; Pedotti, M.; Motteran, L.; Molla, G.; Pollegioni, L.
FAD binding in glycine oxidase from Bacillus subtilis
Biochimie
91
1499-1508
2009
Bacillus subtilis (O31616), Bacillus subtilis
Pedotti, M.; Ghisla, S.; Motteran, L.; Molla, G.; Pollegioni, L.
Catalytic and redox properties of glycine oxidase from Bacillus subtilis
Biochimie
91
604-612
2009
Bacillus subtilis (O31616), Bacillus subtilis
Pedotti, M.; Rosini, E.; Molla, G.; Moschetti, T.; Savino, C.; Vallone, B.; Pollegioni, L.
Glyphosate resistance by engineering the flavoenzyme glycine oxidase
J. Biol. Chem.
284
36415-36423
2009
Bacillus subtilis (O31616), Bacillus subtilis
Jamil, F.; Afza Gardner, Q.T.; Bashir, Q.; Rashid, N.; Akhtar, M.
Mechanistic and stereochemical studies of glycine oxidase from Bacillus subtilis strain R5
Biochemistry
49
7377-7383
2010
Bacillus subtilis, Bacillus subtilis R5
Jamil, F.; Rashid, N.; Gardner, Q.; Akhtar, M.
Gene cloning and characterization of glycine oxidase from newly isolated Bacillus subtilis strain R5
Biologia
66
1-7
2011
Bacillus subtilis (D7UTZ8), Bacillus subtilis R5 (D7UTZ8)
-
Rosini, E.; Piubelli, L.; Molla, G.; Frattini, L.; Valentino, M.; Varriale, A.; DAuria, S.; Pollegioni, L.
Novel biosensors based on optimized glycine oxidase
FEBS J.
281
3460-3472
2014
Bacillus subtilis (O31616), Bacillus subtilis, Bacillus subtilis 168 (O31616)
Nicolia, A.; Ferradini, N.; Molla, G.; Biagetti, E.; Pollegioni, L.; Veronesi, F.; Rosellini, D.
Expression of an evolved engineered variant of a bacterial glycine oxidase leads to glyphosate resistance in alfalfa
J. Biotechnol.
184
201-208
2014
Bacillus subtilis (O31616), Bacillus subtilis, Bacillus subtilis 168 (O31616)
Tatsumi, M.; Hoshino, W.; Kodama, Y.; Ueatrongchit, T.; Takahashi, K.; Yamaguchi, H.; Tagami, U.; Miyano, H.; Asano, Y.; Mizukoshi, T.
Development of a rapid and simple glycine analysis method using a stable glycine oxidase mutant
Anal. Biochem.
587
113447
2019
Bacillus subtilis (O31616), Bacillus subtilis, Bacillus subtilis 168 (O31616)
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