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Enzyme Nomenclature
Information on EC 4.3.1.1 - aspartate ammonia-lyase
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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria
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EC NUMBER 
COMMENTARY 
4.3.1.1
-
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RECOMMENDED NAME
GeneOntology No.
aspartate ammonia-lyase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
L-aspartate = fumarate + NH3
reaction favors aspartate formation
-
-
-
L-aspartate = fumarate + NH3
mechanism
-
L-aspartate = fumarate + NH3
the catalytic mechanism is studied by using a combined quantum-mechanical/molecular-mechanical (QM/MM) approach. Two elementary steps are calculated: The first step is the abstraction of Cbeta proton of L-aspartate by Ser318, which is calculated to be rate limiting. The second step is the cleavage of Calpha- N bond of L-aspartate to form fumarate and ammonia. Ser318 functions as the catalytic base, whereas His188 is a dispensable residue, but its protonation state can influence the active site structure
-
L-aspartate = fumarate + NH3
mechanism; the catalytic mechanism is studied by using a combined quantum-mechanical/molecular-mechanical (QM/MM) approach. Two elementary steps are calculated: The first step is the abstraction of Cbeta proton of L-aspartate by Ser318, which is calculated to be rate limiting. The second step is the cleavage of Calpha- N bond of L-aspartate to form fumarate and ammonia. Ser318 functions as the catalytic base, whereas His188 is a dispensable residue, but its protonation state can influence the active site structure
Bacillus sp. (in: Bacteria) YM55-1
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-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
beta-elimination
enzyme directly forms fumarate by beta-elimination of ammonia
C-N bond cleavage
Deamination
elimination
-
-
of NH3, C-N bond cleavage
-
C-N bond cleavage
oxidative and non-oxidative catabolic mechanism exists
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
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SYSTEMATIC NAME
IUBMB Comments
L-aspartate ammonia-lyase (fumarate-forming)
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CAS REGISTRY NUMBER
COMMENTARY
9027-30-9
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Bacterium cadaveris
Cj0087; zoonotic pathogen, a number of avian species are reservoirs for this organism
UniProt
-
5997, 5998, 6000, 6001, 6002, 6003, 6004, 6005, 6006, 6008, 6009, 6011, 6014, 6016, 6019, 6021, 6022, 6026, 6030, 649266, 649545, 649554, 652275, 657547, 663872, 665790
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-
enzootic (pestoides) isolates A-D; enzootic (pestoides) isolates E, F, G and I; strains Angola and A16
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-
epidemic strains Camel, CO92, KIM10, Kuma, Pestoides J, UG05-0454 /enzootic strains A16, Angola, Pestoides A, Pestoides B, Pestoides C, Pestoides D, Pestoides E, Pestoides F, Pestoides G, Pestoides I
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
-
AspA increases acid survival by producing ammonia. Addition of aspartate increases acid survival of the wild type but not the AspA knockout mutant
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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
hydrazine + fumarate
2-hydrazinosuccinic acid
-
reverse reaction, 100% conversion after 1 day at pH 7
-
-
r
hydroxylamine + fumarate
N-hydroxyaspartic acid
-
reverse reaction, 100% conversion after 20 min at pH 7
-
-
r
L-aspartic acid alpha-amide
(2E)-4-amino-4-oxobut-2-enoate + NH3
-
catalyzed by mutant enzyme K327N, no activity with wild-type enzyme
-
-
?
methoxylamine + fumarate
N-methoxyaspartic acid
-
reverse reaction, 100% conversion after 6 days at pH 8 and after 12 days at pH 7
-
-
r
methylamine + fumarate
N-methylaspartic acid
-
reverse reaction, 100% conversion after 7 day at pH 8
-
-
r
L-aspartate
fumarate + NH3
-
key role in nitrogen metabolism by catalyzing the reversible elimination of NH3 from l-aspartate
-
-
r
L-aspartate
fumarate + NH3
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natural substrate
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-
L-aspartate
fumarate + NH3
-
-
fumarate is a component of the tricarboxylic acid (TCA) cycle
-
?
L-aspartate
fumarate + NH3
-
-
fumarate is a component of the tricarboxylic acid (TCA) cycle
-
?
additional information
?
-
-
important role in the bacterial nitrogen metabolism
-
-
-
additional information
?
-
Bacillus sp. (in: Bacteria) YM55-1
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important role in the bacterial nitrogen metabolism
-
-
-
additional information
?
-
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AspA is one of the most specific enzymes known, no other substrates can replace L-aspartic acid in the deamination reaction
-
-
-
additional information
?
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AspB efficiently processes small substituted amines, but displays very low (methoxylamine and methamine) or no (ethylamine, glycine, and formamide) activity with larger amine nucleophiles
-
-
-
additional information
?
-
-
aspartate enhances oxygen-limited strain growth via fumarate respiration in an aspA-dependent manner
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-
-
additional information
?
-
aspartate enhances oxygen-limited strain growth via fumarate respiration in an aspA-dependent manner
-
-
-
additional information
?
-
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both wild-type and AspA mutant Campylobacter jejuni strains show similar fumarate-dependent growth
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-
-
additional information
?
-
both wild-type and AspA mutant Campylobacter jejuni strains show similar fumarate-dependent growth
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-
-
additional information
?
-
-
no activity with D-aspartic acid or crotonic acid, overview substrate specificity
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?
additional information
?
-
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L-aspartate ammonia-lyase and fumarate hydratase share extensive sequence homology
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-
-
additional information
?
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L-aspartate ammonia-lyase and fumarate hydratase share extensive sequence homology
-
-
-
additional information
?
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-
enzyme synthesis is subject to catabolite repression by glucose and is suppressed under aerobic conditions
-
-
-
additional information
?
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enzyme is essential for the catabolism of the glutamate
-
-
-
additional information
?
-
-
aspartase, fumarase and argininosuccinase are structurally-related enzymes
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-
-
additional information
?
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-
one of the enzymes of the aspartate metabolism in lactic acid bacteria
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-
-
additional information
?
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one of the enzymes of the aspartate metabolism in lactic acid bacteria
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-
additional information
?
-
-
key enzyme involved in the metabolism of aspartate
-
-
-
additional information
?
-
-
L-aspartate ammonia-lyase and fumarate hydratase share extensive sequence homology
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-
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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
L-aspartate
fumarate + NH3
-
key role in nitrogen metabolism by catalyzing the reversible elimination of NH3 from l-aspartate
-
-
r
L-aspartate
fumarate + NH3
-
-
fumarate is a component of the tricarboxylic acid (TCA) cycle
-
?
L-aspartate
fumarate + NH3
-
-
fumarate is a component of the tricarboxylic acid (TCA) cycle
-
?
additional information
?
-
-
important role in the bacterial nitrogen metabolism
-
-
-
additional information
?
-
Bacillus sp. (in: Bacteria) YM55-1
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important role in the bacterial nitrogen metabolism
-
-
-
additional information
?
-
-
AspA is one of the most specific enzymes known, no other substrates can replace L-aspartic acid in the deamination reaction
-
-
-
additional information
?
-
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aspartate enhances oxygen-limited strain growth via fumarate respiration in an aspA-dependent manner
-
-
-
additional information
?
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Q0PC50
aspartate enhances oxygen-limited strain growth via fumarate respiration in an aspA-dependent manner
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-
-
additional information
?
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both wild-type and AspA mutant Campylobacter jejuni strains show similar fumarate-dependent growth
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-
-
additional information
?
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Q0PC50
both wild-type and AspA mutant Campylobacter jejuni strains show similar fumarate-dependent growth
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-
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additional information
?
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L-aspartate ammonia-lyase and fumarate hydratase share extensive sequence homology
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-
-
additional information
?
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L-aspartate ammonia-lyase and fumarate hydratase share extensive sequence homology
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-
-
additional information
?
-
-
enzyme synthesis is subject to catabolite repression by glucose and is suppressed under aerobic conditions
-
-
-
additional information
?
-
-
enzyme is essential for the catabolism of the glutamate
-
-
-
additional information
?
-
-
aspartase, fumarase and argininosuccinase are structurally-related enzymes
-
-
-
additional information
?
-
-
one of the enzymes of the aspartate metabolism in lactic acid bacteria
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-
-
additional information
?
-
-
one of the enzymes of the aspartate metabolism in lactic acid bacteria
-
-
-
additional information
?
-
-
key enzyme involved in the metabolism of aspartate
-
-
-
additional information
?
-
-
L-aspartate ammonia-lyase and fumarate hydratase share extensive sequence homology
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-
-
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ca2+
Cd2+
Mg2+
Mn2+
Zn2+
additional information
Mg2+
-
enzyme is allosterically activated by its substrate and Mg2+ ions, which are required for activity at alkaline pH
Mg2+
aspartase activity is stimulated about 2fold by inclusion of 6 mM magnesium chloride in the assay
Mg2+
-
maximal activity: 1.0 mM for native enzyme, 2.2 mM for monomeric mutant enzyme maspase 1, 1.8 mM for dimeric mutant enzyme maspase 2, 1.6 mM for tetrameric mutant enzyme maspase 3
additional information
-
absolute requirement for divalent metal ion at high pH value
additional information
-
absolute requirement for divalent metal ion at higher pH
additional information
-
no activation by Ca2+, minimal activation by Co2+, pH-dependent activation by divalent cations at high pH values
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Diethylpyrocarbonate
-
inactivation, reactivation with NH2OH, aspartate, fumarate and chloride protect
guanidine hydrochloride
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at concentrations lower than 1 M, activity is gradually decreased suggesting the existence of the native tetrameric form with denaturation intermediates such as dimeric and monomeric forms of the enzyme. At higher concentrations above 1 M, the enzyme completely lost the activity, suggesting that the enzyme structure is completely denatured
MgCl2
-
inhibitor at high concentrations (above 10 mM)
aspartate beta-semialdehyde
-
i.e. aspartic beta-semialdehyde.Inactivates as an active-site directed agent
fumarate
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50% inhibition at a concentration of 0.2 to 0.6 mM, pH 6.2, 50% inhibition at a concentration of 1.1 to 2.5 mM, pH 7.5
KCl
aspartase activity is severely inhibited by potassium chloride
additional information
enzyme is upregulated by oxygen limitation
-
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
AMP
-
allosteric activation, restores original activity of trypsin-treated enzyme
GTP
-
allosteric activation, restores original activity of trypsin-treated enzyme
alpha-methyl-DL-aspartate
-
activates wild-type enzyme, mutant C-terminal-truncated and acetylated enzyme
Trypsin
-
enzyme is activated several-fold by limited treatment; mechanism of activation
-
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
213
fumarate
-
co-substrate: NH3, pH 7.5, 37°, free-enzyme; co-substrate: NH3, pH 7.5, 37°, LentiKats-immobilized enzyme
244
fumarate
-
co-substrate: NH3, pH 7.5, 37°, EupergitC-immobilized enzyme; co-substrate: NH3, pH 7.5, 37°, MANA-agarose-immobilized enzyme
1.54
hydrazine
-
reverse reaction, 750 mM hydrazine, at pH 8, 22°C in 50 mM phosphate
308
hydrazine
-
reverse reaction, 20 mM fumarate, at pH 8, 22°C in 50 mM phosphate
2.78
hydroxylamine
-
reverse reaction, 400 mM hydroxylamine at pH 8, 22°C in 50 mM phosphate
151
hydroxylamine
-
reverse reaction, 20 mM fumarate, at pH 8, 22°C in 50 mM phosphate
9.1
L-aspartate
-
100 mM Bis-Tris-propane hydrochloride buffer, pH 6.2
10
L-aspartate
pH 6.5, recombinant produced and purified AspA enzyme
12.8
L-aspartate
pH 7.0, recombinant produced and purified AspA enzyme
15
L-aspartate
-
his-tagged protein, pH 8.5, 25°C, in 50 mM NaHPO4 buffer
38.2
L-aspartate
pH 8.0, recombinant produced and purified AspA enzyme
1.61
NH3
-
reverse reaction, 200 mM NH3, at pH 8, 22°C in 50 mM phosphate
85
NH3
-
reverse reaction, 20 mM fumarate, at pH 8, 22°C in 50 mM phosphate
additional information
L-aspartate
-
above 1 M, N142Q mutant protein; above 1 M, N326A mutant protein; above 1 M, N326Q mutant protein; above 1 M, T101A mutant protein; above 1 M, T101S mutant protein; above 1 M, T187A mutant protein; above 400 mM, S140A mutant protein; above 400 mM, T141K mutant protein
additional information
additional information
-
enzyme processes L-aspartic acid, but not D-aspartic acid
-
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
92
hydrazine
-
reverse reaction, 750 mM hydrazine, at pH 8, 22°C in 50 mM phosphate
94
hydrazine
-
reverse reaction, 20 mM fumarate, at pH 8, 22°C in 50 mM phosphate
31
hydroxylamine
-
reverse reaction, 400 mM hydroxylamine at pH 8, 22°C in 50 mM phosphate
99
hydroxylamine
-
reverse reaction, 20 mM fumarate, at pH 8, 22°C in 50 mM phosphate
40
L-aspartate
-
his-tagged protein, pH 8.5, 25°C, in 50 mM NaHPO4 buffer
89
NH3
-
reverse reaction, 20 mM fumarate, at pH 8, 22°C in 50 mM phosphate
additional information
L-aspartate
-
above 0.016 1/sec, N326Q mutant protein; above 0.027 1/sec, T141K mutant protein; above 0.12 1/sec, N326A mutant protein; above 0.38 1/sec, T101A mutant protein; above 0.43 1/sec, T187A mutant protein; above 0.88 1/sec, N142Q mutant protein; above 34 1/sec, T101S mutant protein; above 40 1/sec, S140A mutant protein; below 0.001 1/sec, H188A, H188R and H188K mutant protein; below 0.001 1/sec, K324A mutant protein; below 0.001 1/sec, K324D mutant protein; below 0.001 1/sec, K324H mutant protein; below 0.001 1/sec, K324R mutant protein; below 0.001 1/sec, K324S mutant protein; below 0.001 1/sec, K324V mutant protein; below 0.001 1/sec, N142A mutant protein; below 0.001 1/sec, S140G/T141G mutant protein; below 0.001 1/sec, S140K mutant protein; below 0.001 1/sec, S140K/T141K mutant protein; below 0.001 1/sec, S140R mutant protein; below 0.001 1/sec, T141R mutant protein; below 0.001 1/sec, T141V mutant protein
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.044
-
pYV- (lacking low-calcium response plasmid into pseudogenes), 4 mM Ca2+, 250 mmol Tris/HCl buffer, 5.0 mmol MgCl2
0.05
0.06
-
pYV+ (containing low-calcium response plasmid into pseudogenes), 4 mM Ca2+, 250 mmol Tris/HCl buffer, 5.0 mmol MgCl2
0.075
-
pYV+ (containing low-calcium response plasmid into pseudogenes), 250 mmol Tris/HCl buffer, 5.0 mmol MgCl2
0.2
-
mutant L363V, 50 mM HEPES, 50 mM HEPES, 10 mM Mg acetate, 20 mM L-aspartate; mutant Y146D, 50 mM HEPES, 10 mM Mg acetate, 20 mM L-aspartate; recombinant enzyme, 50 mM HEPES, 10 mM Mg acetate, 20 mM L-aspartate
2.06
wild-type aspartase activity, spectrophotometric assay in cell-free extracts from cells grown to early stationary phase in BHI-FCS medium
72
-
double mutant L363V/Y146D, 50 mM HEPES, 10 mM Mg acetate, 20 mM L-aspartate
additional information
0.05
-
pYV- (lacking low-calcium response plasmid into pseudogenes), 250 mmol Tris/HCl buffer, 5.0 mmol MgCl2
additional information
-
AspB is immobilized by covalent attachment on Eupergit (epoxy support) and MANAagarose (amino support), and entrapment in LentiKats (polyvinyl alcohol) with retained activities of 24, 85 and 63%, respectively
additional information
-
His6-tag at enzyme does not affect AspB activity; no activity with D-aspartic acid, L-cysteine, L-histidine, L-phenylalanine, L-glutamine, L-tyrosine, L-serine, L-alanine, L-valine, L-leucine, L-threonine, L-lysine, alpha-methyl-DL-aspartic acid, beta-methyl-DL-aspartic acid, L-glutamate, beta-alanine, beta-DL-aminobutyric acid, beta-asparagine, beta-phenylalanine and beta-leucine as substrates (25 mm substrate in 100 mm Na2HPO4 buffer, pH 9.0 at 37°C)
additional information
-
AspA has a role in the persistence of Campylobacter jejuni in the avian intestine; aspA mutant strain is totally deficient in aspartase enzyme activity, whereas in the wild-type enzyme, an extremely high specific activity is present in stationary-phase cell-free extracts (more than 2 mmol fumarate are produced per min per mg protein)
additional information
AspA has a role in the persistence of Campylobacter jejuni in the avian intestine; aspA mutant strain is totally deficient in aspartase enzyme activity, whereas in the wild-type enzyme, an extremely high specific activity is present in stationary-phase cell-free extracts (more than 2 mmol fumarate are produced per min per mg protein)
additional information
-
0.202 - 0.207 micromol/min/mg in cell-free extracts, value depends on the strain
additional information
-
activity below 0.001, pCD+ (containing low-calcium response plasmid into pseudogenes), 250 mmol Tris/HCl buffer, 5.0 mmol MgCl2; activity below 0.001, pCD+ (containing low-calcium response plasmid into pseudogenes), 4 mM Ca2+; activity below 0.001, pCD- (lacking low-calcium response plasmid into pseudogenes); activity below 0.001, pCD- (lacking low-calcium response plasmid into pseudogenes), 4 mM Ca2+
additional information
-
full AspA activity occurs in pestoides isolates where valine (pestoides A, B, C and D) or serine (pestoides E, F, G and I) occupy position 363. Reduced activity occurs in strains Angola and A16, which contains phenylalanine at position 363. The kcat but not Km of purified AspA from strain Angola is significantly reduced.; full AspA activity occurs in pestoides isolates where valine (pestoides A, B, C and D) or serine (pestoides E, F, G and I) occupy position 363. Reduced activity occurs in strains Angola and A16, which contains phenylalanine at this position. The kcat but not Km of purified AspA from strain Angola is significantly reduced.
additional information
-
below 0.001 micromol/min/mg in cell-free extracts of epidemic strains (Leu in position 363) /0,02-0.008 micromol/min/mg in cell-free extracts of strain A16 and Angola (Phe in position 363), value depends on the strain /0,028-0.064 micromol/min/mg in cell-free extracts of strain Pestoides A-D (Val in position 363), value depends on the strain /0,039-0.052 micromol/min/mg in cell-free extracts of strain Pestoides D-G and Pestoides I (Ser in position 363), value depends on the strain
additional information
-
0.059 - 0.172 micromol/min/mg in cell-free extracts, value depends on the strain
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5
8.2
8.5
8.8
additional information
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7 - 9
-
pH 7.0: about 40% of maximal activity, pH 9.0: about 90% of maximal activity, substrate: L-aspartate, wild-type enzyme
7.5 - 9
-
pH 7.5: about 70% of maximal activity, pH 9.0: about 85% of maximal activity, substrate: L-aspartic acid, mutant enzyme K327N
additional information
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30
37
45
55
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
10 - 35
-
no reactivation between 0-10°C, linear increase at 10-25°C, with a level off between 25-35°C and completely stopped at 35°C
additional information
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
-
strain KIM, possesses a base transversion (GC-TA) at position 146 of AspA, causing replacement of aspartate by tyrosine
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PDB
SCOP
CATH
UNIPROT
ORGANISM
Bacillus sp. YM55-1;
Brucella abortus (strain 2308);
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
48500
50000
51000
52000
52470
-
mass of monomer, determined by an ESI-qTOF mass spectrometer
55000
56000
-
1 * 56000, mutant enzyme maspase 1, SDS-PAGE; 2 * 56000, mutant enzyme maspase 2, SDS-PAGE; 4 * 56000, mutant enzyme maspase 3, SDS-PAGE
193000
200000
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
homotetramer
monomer
tetramer
additional information
homotetramer
-
predicted and determined by dynamic light scattering (DLS), loss of activity in mutants reflects significant changes in subunit association.
tetramer
Bacillus sp. (in: Bacteria) YM55-1
-
4 * 51000, SDS-PAGE; 4 * 51630, deduced from gene sequence
-
tetramer
-
4 * 51218, calculation from sequence of amino acid; 4 * 55000, SDS-PAGE
tetramer
-
4 * 51218, calculation from sequence of amino acid; 4 * 55000, SDS-PAGE
-
tetramer
-
4 * 50000, SDS-PAGE; 4 * 50859, calculation from sequence of amino acid
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Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
in an unliganded state and in complex with L-aspartate at 2.4 and 2.6 A resolution, respectively. AspB forces the bound substrate to adopt a high-energy, enediolate-like conformation that is stabilized, in part, by an extensive network of hydrogen bonds between residues Thr101, Ser140, Thr141, and Ser319 and the substrate's beta-carboxylate group. Substrate binding induces a large conformational change in the SS loop, residuesG317SSIMPGKVN326, from an open conformation to one that closes over the active site. In the closed conformation, the strictly conserved SS loop residue Ser318 is at a suitable position to act as a catalytic base, abstracting the Cbeta proton of the substrate in the first step of the reaction mechanism. The small C-terminal domain of AspB plays an important role in controlling the conformation of the SS loop
-
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
50
55
additional information
50
-
83% loss of activity (wild-type), 70% loss of activity (mutant enzyme) after 45 min
50
-
30 min, 17% residual activity, wild type enzyme; 30 min, 80% residual activity, monomeric enzyme variant
50
-
30 min, native enzyme loses 83% of initial activity, monomeric mutant enzyme maspase 1 loses 23% of initial activity, dimeric mutant enzyme maspase 2 loses 48% of initial activity, tetrameric mutant enzyme maspase 3 loses 59% of initial activity
55
-
20% loss of activity after 60 min; 60 min, 80% residual activity
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
after incubation in the presence of guanidine hydrochloride at a concentration of 1.1 M 50% loss of activity after 60 min
-
after incubation in the presence of guanidine hydrochloride at a concentration of 1.1 M complete inactivation
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enzyme is fairly stable in the presence of high concentrations of ammonium sulfate, potassium phosphate, and KCl
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
over 24 h of microaerobic batch growth, the AspA specific enzyme activity increases 10fold and the relative abundance of the protein increases over 13fold. After transferring initially microaerobic cultures to oxygenlimiting conditions in the absence of an added electron acceptor, there is an immediate cessation of growth but an approximately 3fold rise in AspA specific activity up to 24 h correlated with a smaller increase in the abundance of the protein.
694233
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
2-mercaptoethanol and dithiothreitol protect enzyme against inactivation during storage
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4C, in the presence of various salts thiol compounds, or glycerol stable for 2 weeks
-
4°C, loss of activity after 1 month independently of the presence of glycerol or MgCl2
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enzyme activity can be maintained by freezing up to 6 months in the ammonium sulfate precipitate
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the catalytic function of the purified enzyme which stored at -70°C until use remains stable for at least 1 month at 4°C without appreciable loss of enzymatic activity
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additional information
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
After overexpression of the aspA gene in Escherichia coli BL21, the gene product is purified to homogeneity by ion-exchange and hydrophobic interaction chromatography
by using a combination of diethylaminoethyl cellulose, Red A-agarose,and Sepharose 6B chromatography
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partially purified by heat precipitation and saturation with ammonium sulfate
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recombinant enzyme containing a C-terminal His6 tag is purified by one-step affinity purification
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transformed cells of Escherichia coli JRG1476 are used to prepare the aspA products: proteins are fractionated in buffer with a NaCl gradient on successive anion-exchange Sepharose XL high-capacity and Source 30Q high-resolution chromatography columns
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transformed cells of Escherichia coli JRG1476 are used to prepare the aspA products: proteins are fractionated in purification buffer with a NaCl gradient on successive anion-exchange Sepharose XL high-capacity and Source 30Q high-resolution chromatography columns
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
An aspartase mutant 81-176 unable to utilize any amino acid except serine (defective in microaerobic growth on multiple amino acids) and an aspA sdaA double-mutant (also lacking serine dehydratase) is cloned. The aspA gene is cloned into the pET101 expression vector and overexpressed in Escherichia coli BL21
expression of His-tagged wild-type enzyme and His-tagged mutant enzyme K327N in Escherichia coli
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functional and inactive aspA are cloned and expressed in AspA-deficient Escherichia coli
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functional and inactive aspA of Yersinia pestis KIM is cloned and expressed in AspA-deficient Escherichia coli
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
H188A
-
mutation of His188 to Ala only changes the active site structure and slightly elongates the distance of Cbeta proton of substrate with Ser318, causing the enzyme to remain significant but reduced activity
H188A
Bacillus sp. (in: Bacteria) YM55-1
-
mutation of His188 to Ala only changes the active site structure and slightly elongates the distance of Cbeta proton of substrate with Ser318, causing the enzyme to remain significant but reduced activity
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S140G/T141G
-
implicated in binding the C4 carboxylate group of substrate
S140K/T141K
-
implicated in binding the C4 carboxylate group of substrate
K140I
-
Km value 10fold higher than wild type, comparable increase in Ki for competitive inhibitors
K327N
-
mutant enzyme catalyzes the deamination of L-aspartic acid alpha-amide, 13.5fold increase in Km-value for L-aspartate compared to wild-type value
K55R
-
completeley inactive and insoluble protein, reactivation by an artificial chaperone system including beta-cyclodextrin and cetyltrimethylammonium bromide
V363L
-
mutation in primary structure causing dramatic differences in catalytic activity do not promote significant changes in secondary structure
additional information
additional information
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mutant N217K/T233R/V367G through enzyme modification by direct evolution in order to enhance enzymic activity
additional information
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design of enzyme variants by ligation of subdomains with a random hexapeptide loop, variant with highest activity is a monomer with high thermotolerance
additional information
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construction of hybrid enzyme from alpha-aspartyl dipeptidase and L-aspartase. The hybrid enzyme can be used in synthesis of the precursor for aspartame
additional information
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insertion of a 15-peptide random peptide into the three domains of L-aspartase to enhance their mobility. After directed screening, the three isoforms of monomeric, dimeric and tetrameric enzyme (named maspase 1, maspase 2 and maspase 3) with the activity of L-aspartase are obtained
additional information
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insertion of a 15-peptide random peptide into the three domains of L-aspartase to enhance their mobility. After directed screening, the three isoforms of monomeric, dimeric and tetrameric enzyme (named maspase 1, maspase 2 and maspase 3) with the activity of L-aspartase are obtained
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Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
100% activity recovery is observed when the enzyme is renatured by dilution at concentrations below 1 M guanidine hydrochloride, the renaturation yield at concentrations above 1 M is 40%. The dissociation process from native tetramer to dimer is reversible but the dissociation process from dimer to monomer is not reversible.
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denaturation by addition of high concentrations of guanidine-HCl leads to reversible dissociation and reassembly of the tetrameric structure
reactivation of trypsin activated and native enzyme by dilution after inactivation by 4 M guanidine-HCl
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renaturation by dialysis against 50 mM Tris-HCl buffer ,pH 8.0, at 4C for 2 days
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reversible denaturation is influenced by various environmental factors
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denaturation by addition of high concentrations of guanidine-HCl leads to reversible dissociation and reassembly of the tetrameric structure
-
denaturation by addition of high concentrations of guanidine-HCl leads to reversible dissociation and reassembly of the tetrameric structure
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
analysis
-
scaled-down method for determination of aspartase activity in a 96-well microtitre plate
biotechnology
food industry
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propionic acid bacteria isolates originating from cheese show a wide range of aspartase activity. Aspartase activity is strain-dependent and each strain must be tested separately in order to be able to choose the most suitable starter culture for cheese production.70% of the 100 isolates tested, show very low levels of aspartate activity
medicine
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involvement of enzyme in blood clotting and activation of plasminogen, overview
synthesis
biotechnology
-
enhancement of recombinant protein production in Escherichia coli by coproduction of aspartase. The excretion of acetate by the aerobic growth of Escherichia coli on glucose is a manifestation of imbalanced flux between glycolysis and the tricarboxylic acid (TCA) cycle. This may restrict the production of recombinant proteins in E. coli, due to the limited amounts of precursor metabolites produced in TCA cycle. To approach this issue, an extra supply of intermediate metabolites in TCA cycle is made by conversion of aspartate to fumarate, a reaction mediated by the activity of L-aspartate ammonia-lyase
biotechnology
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putative and attractive enzyme for the enantioselective synthesis of N-substituted aspartic acids
biotechnology
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a complete biocatalytic process to synthesize high concentrations of L-aspartate catalyzed by aspartase from Bacillus sp. YM55-1 (AspB) is established using an immobilized enzyme in three different supports. MANA-agarose derivative could be selected as the most suitable biocatalyst for the synthesis of Asp due to the simplicity of the method and performance
biotechnology
Bacillus sp. (in: Bacteria) YM55-1
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a complete biocatalytic process to synthesize high concentrations of L-aspartate catalyzed by aspartase from Bacillus sp. YM55-1 (AspB) is established using an immobilized enzyme in three different supports. MANA-agarose derivative could be selected as the most suitable biocatalyst for the synthesis of Asp due to the simplicity of the method and performance
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synthesis
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industrial production of L-aspartic acid using polyurethane-immobilized cells containing aspartase
synthesis
-
enzyme or whole cells are used for the industrial production of L-aspartate, which together with L-phenylalanine is the basis of the sweetener aspartame
synthesis
-
production of L-aspartic acid, which is used as an intermediate for aspartame, an artificial sweetener. It is also used as acidulant in pharmaceuticals and foods
synthesis
-
production of L-aspartic acid, which is used as a precursor for the synthesis of the low calorie synthetic sweetener aspartame
synthesis
-
construction of hybrid enzyme from alpha-aspartyl dipeptidase and L-aspartase. The hybrid enzyme can be used in synthesis of the precursor for aspartame. Synthesizing aspartate from fumarate and NH4+, and then taking advantage of the catalytic action of alpha-aspartyl dipeptidase, the precursor of aspartame is produced
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