Information on EC 1.4.3.16 - L-aspartate oxidase

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

EC NUMBER
COMMENTARY hide
1.4.3.16
-
RECOMMENDED NAME
GeneOntology No.
L-aspartate oxidase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
L-aspartate + O2 = iminosuccinate + H2O2
show the reaction diagram
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-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
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oxidative deamination
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-
-
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redox reaction
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-
-
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reduction
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
superpathway of nicotine biosynthesis
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nicotine biosynthesis
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NAD biosynthesis I (from aspartate)
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NAD metabolism
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Alanine, aspartate and glutamate metabolism
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Nicotinate and nicotinamide metabolism
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Metabolic pathways
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SYSTEMATIC NAME
IUBMB Comments
L-aspartate:oxygen oxidoreductase
A flavoprotein (FAD). L-Aspartate oxidase catalyses the first step in the de novo biosynthesis of NAD+ in some bacteria. O2 can be replaced by fumarate as electron acceptor, yielding succinate [5]. The ability of the enzyme to use both O2 and fumarate in cofactor reoxidation enables it to function under both aerobic and anaerobic conditions [5]. Iminosuccinate can either be hydrolysed to form oxaloacetate and NH3 or can be used by EC 2.5.1.72, quinolinate synthase, in the production of quinolinate. The enzyme is a member of the succinate dehydrogenase/fumarate-reductase family of enzymes [5].
CAS REGISTRY NUMBER
COMMENTARY hide
69106-47-4
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
UniProt
Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
OT-3
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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although the enzyme mutation dramatically affects NADPH oxidase RBOHD function, it does not affect functions carried out by other members of the RBOH family, such as RBOHC and RBOHF
metabolism
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the enzyme catalyzes the first irreversible step in the de novo biosynthesis of NAD+
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
3-hydroxy-erythro-L-aspartate + O2
2-amino-3-hydroxy-2-butenedioic acid + H2O2
show the reaction diagram
-
-
-
-
?
L-asparagine + H2O + O2
4-amino-2,4-dioxobutanoate + NH3 + H2O2
show the reaction diagram
L-asparagine + O2
4-amino-2-imino-4-oxobutanoate + H2O2
show the reaction diagram
KC333624;, Q972D2;
Vmax/Km is 63fold lower compared to L-aspartate
-
-
?
L-aspartate + fumarate
iminosuccinate + succinate
show the reaction diagram
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-
-
-
?
L-aspartate + H2O + fumarate
oxaloacetate + NH3 + succinate
show the reaction diagram
L-aspartate + H2O + O2
alpha-iminosuccinate + H2O2
show the reaction diagram
-
-
-
-
?
L-aspartate + H2O + O2
oxaloacetate + NH3 + H2O2
show the reaction diagram
L-aspartate + O2
iminosuccinate + H2O2
show the reaction diagram
L-aspartate + O2
oxaloacetate + NH3 + H2O2
show the reaction diagram
-
-
-
?
L-glutamate + H2O + O2
2-oxopentanedioate + NH3 + H2O2
show the reaction diagram
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low activity
-
-
?
N-acetyl-L-aspartate + O2
? + H2O2
show the reaction diagram
-
-
-
-
?
N-formyl-L-aspartate + O2
? + H2O2
show the reaction diagram
-
-
-
-
?
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
L-asparagine + H2O + O2
4-amino-2,4-dioxobutanoate + NH3 + H2O2
show the reaction diagram
L-aspartate + fumarate
iminosuccinate + succinate
show the reaction diagram
-
-
-
-
?
L-aspartate + H2O + fumarate
oxaloacetate + NH3 + succinate
show the reaction diagram
-
it is very likely, that fumarate and not O2 is the physiological electron acceptor in vivo
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-
?
L-aspartate + H2O + O2
alpha-iminosuccinate + H2O2
show the reaction diagram
-
-
-
-
?
L-aspartate + H2O + O2
oxaloacetate + NH3 + H2O2
show the reaction diagram
L-aspartate + O2
iminosuccinate + H2O2
show the reaction diagram
-
-
-
-
?
L-glutamate + H2O + O2
2-oxopentanedioate + NH3 + H2O2
show the reaction diagram
-
low activity
-
-
?
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
D-Aspartate
iodoacetate
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L-asparagine
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L-aspartate
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substrate inactivation
meso-tartrate
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oxaloacetate
KC333624;, Q972D2;
weak inhibition; weak inhibition
Tetranitromethane
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-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-oxoglutarate
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L-aspartate
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substrate activation above 1.0 mM
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4.7
3-hydroxy-erythro-L-aspartate
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pH 8.0, 25°C, recombinant wild-type enzyme
1.43
fumarate
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18.1
L-asparagine
KC333624;, Q972D2;
apparent value, at pH 8.0 and 37°C
0.038 - 75.44
L-aspartate
3.2 - 17
N-acetyl-L-aspartate
3.7 - 17.5
N-formyl-L-aspartate
0.33
O2
KC333624;, Q972D2;
pH 8, 37°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.173
3-hydroxy-erythro-L-aspartate
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pH 8.0, 25°C, recombinant wild-type enzyme
0.5
fumarate
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0.0333 - 23.69
L-aspartate
0.0035 - 0.0055
N-acetyl-L-aspartate
0.0013 - 0.0077
N-formyl-L-aspartate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0368
3-hydroxy-erythro-L-aspartate
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pH 8.0, 25°C, recombinant wild-type enzyme
0.35
fumarate
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0.008 - 5.56
L-aspartate
0.00021 - 0.0017
N-acetyl-L-aspartate
0.00007 - 0.00208
N-formyl-L-aspartate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 8
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about 40% activity at pH 6.0, about 50% activity at pH 8.0
8.8 - 12
KC333624;, Q972D2;
pH 8.8: about 50% of maximal activity, pH 12.0: about 70% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
KC333624;, Q972D2;
assay at
40
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at pH 8.0
60 - 80
KC333624;, Q972D2;
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40 - 95
KC333624;, Q972D2;
40°C: about 50% of maximal activity, 95°C: about 60% of maximal activity
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
UNIPROT
ORGANISM
Escherichia coli (strain K12);
Q8ZMX9
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720);
Q972D2
Sulfolobus tokodaii (strain DSM 16993 / JCM 10545 / NBRC 100140 / 7);
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
47000
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gel filtration
52000
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recombinant enzyme, SDS-PAGE
52580
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calculated from the amino acid sequence
53647
KC333624;, Q972D2;
1 * 53647, calculated from amino acid sequence
55000
monomer, gel filtration
59900
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x * 59900, calculated from amino acid sequence
60280
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sequence analysis
83000
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gel filtration
115000
dimer, gel filtration
151000
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gel filtration
536467
KC333624;, Q972D2;
x * 536467, calculated from sequence
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
gel filtration, 2 * 55000 Da, dimer in the absence of NaCl
monomer
trimer
additional information
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three dimensional structure of holo-wild-type-LASPO, modelling, overview
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
sitting drop vapour diffusion method with 23% (w/v) polyethylene glycol 8000, 0.2 M MgCl2 and 0.1 M Tris/HCl buffer (pH 8.6)
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.7 - 10.3
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when heated at 50 °C for 30 min, the enzyme does not lose activity at pH values from 3.7 to 10.3
685396
7 - 10
KC333624;, Q972D2;
60 min, stable; no significant change in activity is observed up to 400 min of incubation at pH 7.5. More than 70% of the initial activity is recovered after 60 min at 37°C. Below pH 8.0 and above pH 10.0, a significant time-dependent inactivation is apparent
721394
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
KC333624;, Q972D2;
more than 70% of the initial activity is recovered after 60 min at 37°C
40 - 50
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the enzyme is stable for 60 min at up to 40°C, but rapid losses in activity are observed at 50°C after 5 min
83
KC333624;, Q972D2;
Tm-value determinedv by fluoresecence at 340 nm
86
KC333624;, Q972D2;
Tm-value determinedv by fluoresecence at 525 nm
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
stable at 4°C for at least 2 months without loss of activity
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urea, 7 M, denaturation
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°, purified enzyme at pH 7.5, several months, nploss of activity
KC333624;, Q972D2;
4°C, pH 7.5, in the dark, stable for months
KC333624;, Q972D2;
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; HiTrap nickel chelating affinity column chromatography, and Superdex 200 gel filtration
KC333624;, Q972D2;
ammonium sulfate precipitation and Butyl Toyopearl column chromatography
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HiTrap nickel chelating column chromatography and Superdex 75 gel filtration
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overexpression in Escherichia coli
using NH4SO4 precipitation and using a DEAE-sepharose column
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
expressed in Escherichia coli BL21(DE3) cells; expression in Escherichia coli in the active form as holoenzyme
KC333624;, Q972D2;
expressed in Escherichia coli BL21-Gold (DE3) cells
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expressed in Escherichia coli mutants deficient in AO, transformation of the heterozygous plants with a vector that constitutively expresses the wild-type AO protein fused to GFP at the C terminus
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expressed in Escherichia coli strain BL21(DE3) codon plus RIL
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expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E121A
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme, catalytically inactive against either 3-OH-erythro- or 3-OH-threo-L-aspartate, but is active with N-acetyl- and N-formyl-L-aspartate
E121D
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site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme, catalytically inactive against either 3-OH-erythro- or 3-OH-threo-L-aspartate
E121K
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site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme, catalytically inactive against either 3-OH-erythro- or 3-OH-threo-L-aspartate
E121Q
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme, catalytically inactive against either 3-OH-erythro- or 3-OH-threo-L-aspartate
H244A
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binds substrate analogues with higher dissociation constants and presents lower kcat/Km values in the reduction of fumarate
H244S
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binds substrate analogues with higher dissociation constants and presents lower kcat/Km values in the reduction of fumarate
H351A
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binds substrate analogues with higher dissociation constants and presents lower kcat/Km values in the reduction of fumarate
H351S
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binds substrate analogues with higher dissociation constants and presents lower kcat/Km values in the reduction of fumarate
R386L
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binds substrate analogues with higher dissociation constants and presens lower kcat/Km values in the reduction of fumarate
H244A
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the mutant shows reduced catalytic efficiency compared to the wild type enzyme
H351A
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inactive
Q242A
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the mutant shows reduced catalytic efficiency compared to the wild type enzyme
R290A
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the mutant shows reduced catalytic efficiency compared to the wild type enzyme
R386A
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inactive
S389A
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the mutant shows reduced catalytic efficiency compared to the wild type enzyme
T259A
-
inactive
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
KC333624;, Q972D2;
StLASPO represents an appropriate biocatalyst for the resolution of racemic solutions of D,L-aspartate and a well-suited protein scaffold to evolve a L-amino acid oxidase activity by protein engineering
additional information
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is essential for plant growth and development
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