Information on EC 1.1.3.2 - L-lactate oxidase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
1.1.3.2
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RECOMMENDED NAME
GeneOntology No.
L-lactate oxidase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(S)-lactate + O2 = pyruvate + H2O2
show the reaction diagram
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-
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SYSTEMATIC NAME
IUBMB Comments
(S)-lactate:oxygen 2-oxidoreductase
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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D4YFM2
UniProt
Manually annotated by BRENDA team
cf. EC 1.1.3.1, enzyme shows high L-lactate oxidase and low glycolate oxidase activities
UniProt
Manually annotated by BRENDA team
Gram-negative soil bacterium
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-
Manually annotated by BRENDA team
Gram-negative soil bacterium KY6
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-
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Manually annotated by BRENDA team
cf. EC 1.1.3.1, enzyme shows high L-lactate oxidase and low glycolate oxidase activities
UniProt
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(R)-lactate + 2,6-dichlorophenolindophenol
?
show the reaction diagram
(S)-2-hydroxy-2-phenylacetate + O2
phenylpyruvate + H2O2
show the reaction diagram
-
-
-
-
?
(S)-2-hydroxybutanoate + O2
2-oxobutanoate + H2O2
show the reaction diagram
-
-
-
-
?
(S)-lactate + 2,6-dichlorophenolindophenol
pyruvate + ?
show the reaction diagram
(S)-lactate + O2
pyruvate + H2O2
show the reaction diagram
(S)-mandelic acid + O2
phenylpyruvate + H2O2
show the reaction diagram
-
-
-
-
?
2-hydroxy-2-(4-chlorophenyl)acetate + O2
(4-chlorophenyl)pyruvate + H2O2
show the reaction diagram
-
-
-
-
?
2-hydroxy-2-(4-hydroxyphenyl)acetate + O2
(4-hydroxyphenyl)pyruvate + H2O2
show the reaction diagram
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-
-
-
?
2-hydroxy-2-(4-methoxyphenyl)acetate + O2
(4-methoxyphenyl)pyruvate + H2O2
show the reaction diagram
-
-
-
-
?
2-hydroxy-2-(4-methylphenyl)acetate + O2
(4-methylphenyl)pyruvate + H2O2
show the reaction diagram
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-
-
-
?
2-hydroxy-2-(4-nitrophenyl)acetate + O2
(4-nitrophenyl)pyruvate + H2O2
show the reaction diagram
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-
-
-
?
2-hydroxy-2-phenylacetate + O2
phenylpyruvate + H2O2
show the reaction diagram
-
-
-
-
?
2-hydroxybutanoate + O2
2-oxobutanoate + H2O2
show the reaction diagram
-
-
-
-
?
2-hydroxypentanoate + O2
2-oxopentanoate + H2O2
show the reaction diagram
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-
-
-
?
glycerate + O2
hydroxypyruvate + H2O2
show the reaction diagram
glycolate + 2,6-dichlorophenolindophenol
?
show the reaction diagram
glyoxylate + O2
oxalate + H2O2
show the reaction diagram
15.3% of the activity with L-lactate
-
-
?
L-2-hydroxyisocaproate + 2,6-dichlorophenolindophenol
?
show the reaction diagram
L-alpha-hydroxy-beta-methylvalerate + O2
3-methyl-2-oxopentanoate + H2O2
show the reaction diagram
-
-
-
-
?
L-lactate + O2
pyruvate + H2O2
show the reaction diagram
L-malic acid + 2,6-dichlorophenolindophenol
?
show the reaction diagram
Gram-negative soil bacterium
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no decarboxylation
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?
lactate + 2,6-dichlorophenolindophenol
?
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(R)-lactate
alpha-hydroxybutanoate
D4YFM2
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Bromopyruvate
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irreversible inactivation
Cibacron blue 3GA
D4YFM2
at low concentrations, Cibacron Blue 3GA behaves as a dye-, pH- and time-dependent inhibitor, non-linear inhibition
D-lactate
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competitive
DL-2-Hydroxybutanoate
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competitive
FeSO4
Gram-negative soil bacterium
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86% inhibition at 1 mM
glycolate
HgCl2
Gram-negative soil bacterium
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28% inhibition at 1 mM
iodoacetate
Gram-negative soil bacterium
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33% inhibition at 1 mM
L-leucine
Gram-negative soil bacterium
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29% inhibition at 1 mM
L-phenylalanine
Gram-negative soil bacterium
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28% inhibition at 1 mM
Lactate
MnSO4
Gram-negative soil bacterium
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47% inhibition at 1 mM
NAD+
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competitive
oxalate
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competitive
p-chloromercuribenzoate
Gram-negative soil bacterium
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21% inhibition at 1 mM
ZnSO4
Gram-negative soil bacterium
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28% inhibition at 1 mM
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
EDTA
Gram-negative soil bacterium
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40% activation at 1 mM
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.14
(R)-lactate
Gram-negative soil bacterium
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-
0.00034 - 46
(S)-lactate
0.3 - 20
(S)-mandelic acid
18 - 27
2-hydroxybutanoate
5.5 - 10
2-hydroxypentanoate
5 - 53
glycerate
0.039 - 103
L-lactate
0.2
Lactate
Gram-negative soil bacterium
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-
0.024 - 0.16
O2
additional information
additional information
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detailed analysis of reductive and oxidative half-reaction
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
6.6
(S)-2-Hydroxy-2-phenylacetate
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mutant A95G, pH 7.0, 25C
5 - 280
(S)-lactate
0.0033 - 15.8
(S)-mandelic acid
11 - 510
2-hydroxybutanoate
1.25 - 51.7
2-hydroxypentanoate
0.87 - 31.3
glycerate
12
L-alpha-hydroxy-beta-methylvalerate
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mutant A95G, pH 7.0, 25C
250 - 283
L-lactate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
9.61 - 176
(S)-lactate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
9.04
(R)-lactate
D4YFM2
pH 7.0, 37C
42.4
alpha-hydroxybutanoate
D4YFM2
pH 7.0, 37C
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2.9
D-lactate
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pH 7.0, 37C
15
DL-2-Hydroxybutanoate
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pH 7.0, 37C
0.32 - 1.2
glycolate
5.3
oxalate
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pH 7.0, 37C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
114.5
D4YFM2
pH 7.0, 37C
210
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pH 7.0, 37C
272
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pH 7.0, 37C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.7
Gram-negative soil bacterium
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70
Gram-negative soil bacterium
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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intermembrane space. H2O2 is produced independently on the respiratory chain with 1:1 stoichiometry with pyruvate, due to a putative flavin-dependent L-lactate oxidase restricted to the intermembrane space
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45000
Gram-negative soil bacterium
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? * 45000, SDS-PAGE
80000
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gel filtration
162000
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gel filtration
187000
D4YFM2
gel filtration
204000
Gram-negative soil bacterium
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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2 * 40000, SDS-PAGE and calculated
tetramer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
1.9 A crystal structure, Y191F variant bound with FMN and pyruvate. Mutation has a strictly locally disruptive effect
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in complex with pyruvate, to 1.9 A resolution. One pyruvate molecule binds to the active site and locates near the N5 position of FMN for subunits, A, B, and D in the asymmetric unit. The pyruvate molecule is stabilized by the interaction of its carboxylate group with the side-chain atoms of Tyr40, Arg181, His265, and Arg268, and of its keto-oxygen atom with the side-chain atoms of Tyr146, Tyr215, and His265
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mutant A95G in complex with pyruvate, to 1.65 A resolution. Mutation leads to increased steric volume available in the active site
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mutant Y215F in complex with pyruvate, to 2.6 A resolution. The hydrogen bond between the phenolic hydroxyl and the keto oxygen in pyruvate is replaced with a potentially stronger hydrophobic interaction between the phenylalanine and the methyl group of pyruvate. Residues 200 through 215 or 216 appear to be disordered
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native enzyme and its complex with D-lactate at pH 4.5. In the complex structure, the D-lactate resides in the substrate-binding site, but acitve site His265 flips far away from the D-lactate, as compared with its conformation in the unbound state at pH 8.0. The flip of His265 triggers a large structural rearrangement. The reductive half-reaction mechanism may release pyruvate through hydride transfer. In the oxidative half-reaction, His265 flips back, pushing molecular oxygen into the substrate-binding site as the second substrate, and the reverse reaction takes place to produce hydrogen peroxide
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to 2.1 A resolution. LOX crystallizes as two tightly packed tetramers in the asymmetric unit, each having fourfold symmetry. Residues Tyr40, Arg268, His265, Tyr146 and Asp174 are involved in substrate binding
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to 2.1 A resolution. The LOX monomer structure has a typical alpha8/beta8 motif. Five residues around the FMN prosthetic group of LOX, act synergistically to discriminate between the L/D configurations of lactate. Space group I422 with unit-cell parameters a = b = 191.096 A , c = 194.497A and a = b = c = 90 with four monomers per asymmetric unit
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to 3.0 A resolution, space group P212121 with unit cell parameters a = 118.4 A, b = 138.4 A, c = 104.6 A. The enzyme may exist as an octameric form with noncrystallographic two- and four-fold axes in the center of the octamer
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.7 - 9.5
Gram-negative soil bacterium
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stable for 24 h at 4C
440395
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0 - 45
Gram-negative soil bacterium
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stable for 10 min
55
Gram-negative soil bacterium
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50% activity after 10 min
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
expression is induced during growth on L-lactate in the exponential growth phase and by growth on glucose after adaptation to stress conditions
expression is induced upon growth on lactate
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A96L
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engineering the enzyme in order to minimize the effects of oxygen interference on sensor strips. Mutant A96L shows a drastic reduction in oxidase activity using molecular oxygen as the electron acceptor and a small increase in dehydrogenase activity employing an artificial electron acceptor. After immobilization on a screen-printed carbon electrode and under argon or atmospheric conditions, the response current increases linearly from 0.05 to 0.5 mM L-lactate for both wild-type and mutant A96L. Under atmospheric conditions, the response of wild-type electrode is suppressed by 9-12% due to oxygen interference. The mutant maintains 56-69% of the response current at the same L-lactate level and minimizes the relative bias error to -19% from -49% of wild-type
R181K
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mutation in conserved residue, efficiency of reduction of the oxidized flavin by L-lactate is greatly reduced
R181K/R268K
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mutation in conserved residue, efficiency of reduction of the oxidized flavin by L-lactate is greatly reduced
R181M
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mutation in conserved residue, efficiency of reduction of the oxidized flavin by L-lactate is greatly reduced
R268K
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mutation in conserved residue, efficiency of reduction of the oxidized flavin by L-lactate is greatly reduced. Mutation also results in a slow conversion of the 8-CH3-substituent of FMN to yield 8-formyl-FMN
S218C
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introduction of a site for chemical modification, about 50% of wild-type activity
Y191A
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28fold decrease in release of pyruvate, , binding of L-lactate is strongly affected
Y191F
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4.7fold decrease in release of pyruvate
Y191L
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19fold decrease in release of pyruvate, binding of L-lactate is strongly affected
Y215F
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mutation in binding pocket, mutant shows slowed flavin reduction and oxidation by up to 33-fold. Pyruvate release is also decelerated and is the slowest step overall
Y215H Y215F
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mutation in binding pocket, mutant shows slowed flavin reduction and oxidation by up to 33-fold. Pyruvate release is also decelerated
F212V
change in the active site to that of Arabidopsis thaliana glycolate oxidase 2, 25fold decrease in the L-lactate oxidase/glycolate oxidase activity ratio
L112W
change in the active site to that of Arabidopsis thaliana glycolate oxidase 2, 2fold decrease in the L-lactate oxidase/glycolate oxidase activity ratio
M82T
change in the active site to that of Arabidopsis thaliana glycolate oxidase 2, 10fold decrease in the L-lactate oxidase/glycolate oxidase activity ratio
M82T/L112W/F212V
change in the active site to that of Arabidopsis thaliana glycolate oxidase 2, reverse the L-lactate oxidase/glycolate oxidase activity ratio
additional information
gene variant type 2 reveals a 51-nucleotide insertion in LctO, resulting in a 17-amino-acid repeat in the gene product, and formation of an extra loop in the monomeric protein structure. Upon expression in Escherichia coli, the higher-molecular-weight type 2 enzyme exhibits higher activity. Growth rates of Streptococcus iniae expressing the type 2 enzyme are not reduced at lactate concentrations of 0.3% and 0.5%, whereas a strain expressing the type 1 enzyme exhibits reduced growth rates at these lactate concentrations
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
biotechnology
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coupling of mutant S218C in 94% yield to maleimide-activated methoxypoly(ethylene glycol) 5000. PEGylation causes about 30% small decrease in the specific activity of the S218C mutant, and it does not change the protein stability