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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-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
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oxidative deamination
-
-
-
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redox reaction
-
-
-
-
reduction
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Alanine, aspartate and glutamate metabolism
-
-
Metabolic pathways
-
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NAD biosynthesis I (from aspartate)
-
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Nicotinate and nicotinamide metabolism
-
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nicotine biosynthesis
-
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superpathway of nicotine biosynthesis
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NAD metabolism
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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
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
UniProt
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
OT-3
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
-
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
-
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
-
-
-
-
?
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
-
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
-
-
?
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
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
D-Aspartate
iodoacetate
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-
L-asparagine
-
-
L-aspartate
-
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
-
-
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
-
pH 8.0, 25C, recombinant wild-type enzyme
1.43
fumarate
-
18.1
L-asparagine
KC333624, Q972D2
apparent value, at pH 8.0 and 37C
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, 37C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.173
3-hydroxy-erythro-L-aspartate
Escherichia coli
-
pH 8.0, 25C, recombinant wild-type enzyme
0.5
fumarate
Bacillus subtilis
P38032
-
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
Escherichia coli
-
pH 8.0, 25C, recombinant wild-type enzyme
41304
0.35
fumarate
Bacillus subtilis
P38032
-
170
0.008 - 5.56
L-aspartate
97
0.00021 - 0.0017
N-acetyl-L-aspartate
2276
0.00007 - 0.00208
N-formyl-L-aspartate
4544
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
-
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
-
at pH 8.0
60 - 80
KC333624, Q972D2
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40 - 95
KC333624, Q972D2
40C: about 50% of maximal activity, 95C: 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
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)
Sulfolobus tokodaii (strain DSM 16993 / JCM 10545 / NBRC 100140 / 7)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
47000
-
gel filtration
52000
-
recombinant enzyme, SDS-PAGE
52580
-
calculated from the amino acid sequence
53647
KC333624, Q972D2
1 * 53647, calculated from amino acid sequence
55000
monomer, gel filtration
59900
-
x * 59900, calculated from amino acid sequence
60280
-
sequence analysis
83000
-
gel filtration
115000
dimer, gel filtration
151000
-
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
-
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
-
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 37C. 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 37C
40 - 50
-
the enzyme is stable for 60 min at up to 40C, but rapid losses in activity are observed at 50C 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 4C for at least 2 months without loss of activity
-
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
4C, 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
-
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
-
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
-
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
-
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
-
binds substrate analogues with higher dissociation constants and presents lower kcat/Km values in the reduction of fumarate
H244S
-
binds substrate analogues with higher dissociation constants and presents lower kcat/Km values in the reduction of fumarate
H351A
-
binds substrate analogues with higher dissociation constants and presents lower kcat/Km values in the reduction of fumarate
H351S
-
binds substrate analogues with higher dissociation constants and presents lower kcat/Km values in the reduction of fumarate
R386L
-
binds substrate analogues with higher dissociation constants and presens lower kcat/Km values in the reduction of fumarate
H244A
-
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
H351A
-
inactive
Q242A
-
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
R290A
-
the mutant shows reduced catalytic efficiency compared to the wild type enzyme
R386A
-
inactive
S389A
-
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
-
is essential for plant growth and development
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