Information on EC 1.2.1.11 - aspartate-semialdehyde dehydrogenase

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

EC NUMBER
COMMENTARY hide
1.2.1.11
-
RECOMMENDED NAME
GeneOntology No.
aspartate-semialdehyde dehydrogenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
L-aspartate 4-semialdehyde + phosphate + NADP+ = L-4-aspartyl phosphate + NADPH + H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
Phosphorylation
-
-
-
-
redox reaction
reduction
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of antibiotics
-
-
Biosynthesis of secondary metabolites
-
-
Cysteine and methionine metabolism
-
-
ectoine biosynthesis
-
-
Glycine, serine and threonine metabolism
-
-
grixazone biosynthesis
-
-
L-homoserine biosynthesis
-
-
L-lysine biosynthesis I
-
-
L-lysine biosynthesis II
-
-
L-lysine biosynthesis III
-
-
L-lysine biosynthesis VI
-
-
Lysine biosynthesis
-
-
Metabolic pathways
-
-
Microbial metabolism in diverse environments
-
-
Monobactam biosynthesis
-
-
norspermidine biosynthesis
-
-
spermidine biosynthesis II
-
-
threonine metabolism
-
-
SYSTEMATIC NAME
IUBMB Comments
L-aspartate-4-semialdehyde:NADP+ oxidoreductase (phosphorylating)
-
CAS REGISTRY NUMBER
COMMENTARY hide
9000-98-0
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
strain IAM 12477
-
-
Manually annotated by BRENDA team
strain NCIMB 8826
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
wild type strain G165-3A and mutant strain G184-1A
-
-
Manually annotated by BRENDA team
no activity in Homo sapiens
-
-
-
Manually annotated by BRENDA team
strain NRRL 3585, gene asd
SwissProt
Manually annotated by BRENDA team
strain NRRL 3585, gene asd
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
aspartate-4-semialdehyde + phosphate + NADP+
L-4-aspartyl phosphate + NADPH + H+
show the reaction diagram
beta-3-methylaspartyl phosphate + NADPH
beta-3-methylaspartate 4-semialdehyde + phosphate + NADP+
show the reaction diagram
-
-
-
?
L-4-aspartyl phosphate + NADPH
L-aspartate 4-semialdehyde + phosphate + NADP+
show the reaction diagram
L-4-aspartyl phosphate + NADPH
L-aspartate-4-semialdehyde + phosphate + NADP+
show the reaction diagram
L-4-aspartyl phosphate + NADPH + H+
L-aspartate 4-semialdehyde + phosphate + NADP+
show the reaction diagram
L-aspartate 4-semialdehyde + phosphate + NAD+
L-4-aspartyl phosphate + NADH + H+
show the reaction diagram
-
-
-
-
?
L-aspartate 4-semialdehyde + phosphate + NADP+
L-4-aspartyl phosphate + NADPH
show the reaction diagram
L-aspartate 4-semialdehyde + phosphate + NADP+
L-4-aspartyl phosphate + NADPH + H+
show the reaction diagram
L-aspartate-4-semialdehyde + cacodylate + NADP+
L-4-aspartyl cacodylate + NADPH
show the reaction diagram
10% of the activity with phosphate
-
-
r
L-aspartate-4-semialdehyde + phosphate + NADP+
L-4-aspartyl phosphate + NADPH
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
aspartate-4-semialdehyde + phosphate + NADP+
L-4-aspartyl phosphate + NADPH + H+
show the reaction diagram
L-4-aspartyl phosphate + NADPH
L-aspartate 4-semialdehyde + phosphate + NADP+
show the reaction diagram
L-4-aspartyl phosphate + NADPH
L-aspartate-4-semialdehyde + phosphate + NADP+
show the reaction diagram
L-4-aspartyl phosphate + NADPH + H+
L-aspartate 4-semialdehyde + phosphate + NADP+
show the reaction diagram
L-aspartate 4-semialdehyde + phosphate + NADP+
L-4-aspartyl phosphate + NADPH + H+
show the reaction diagram
L-aspartate-4-semialdehyde + phosphate + NADP+
L-4-aspartyl phosphate + NADPH
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NAD+
-
in contrast to Gram-negative bacterial aspartate-beta-semialdehyde dehydrogenases which are specific for NADP+ the enzyme of Gram-positive showed a dual specificity for NAD+ and NADP+
additional information
-
NAD+: 0.1% or less the rate of reaction with NADP+
-
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(1R)-5-[(2-carboxyphenyl)carbamoyl]cyclohexa-3,5-diene-1,3-dicarboxylate
(1R)-5-[(3-nitrophenyl)carbamoyl]cyclohexa-3,5-diene-1,3-dicarboxylate
(1R)-5-[(4-nitrophenyl)carbamoyl]cyclohexa-3,5-diene-1,3-dicarboxylate
(2R,3aR)-5-[(methylsulfonyl)methyl]-2,3,3a,4-tetrahydro-1H-indole-2-carboxylate
(2R,5R)-2,3,4,5-tetrahydropyridine-2,5-dicarboxylate
(2R,5R)-5-nitro-2,3,4,5-tetrahydropyridine-2-carboxylate
(2R,7aR)-2,3,7,7a-tetrahydro-1H-indole-2,6-dicarboxylate
(2R,7aR)-6-hydroxy-2,3,7,7a-tetrahydro-1H-indole-2-carboxylate
(2R,7aR)-6-nitro-2,3,7,7a-tetrahydro-1H-indole-2-carboxylate
(2R,8aR)-2,7-dinitro-1,2,8,8a-tetrahydronaphthalene
(3aR)-2-oxo-2,3,3a,4-tetrahydro-1H-benzimidazole-5-carboxylic acid
(3aR)-5-nitro-1,3,3a,4-tetrahydro-2H-benzimidazol-2-one
(3aR)-5-nitro-3a,4-dihydro-1H-indene-1,3(2H)-dione
(3aR)-5-nitro-3a,4-dihydro-1H-isoindole-1,3(2H)-dione
(3aR)-6-chloro-5-nitro-3a,4-dihydro-1H-isoindole-1,3(2H)-dione
(7aR)-2-oxo-2,3,7,7a-tetrahydro-1H-indole-6-carboxylate
(7aR)-3-(carboxylatomethyl)-6-nitro-7,7a-dihydro-1H-indole-2-carboxylate
(S)-2-amino-5-fluoro-4-oxo-5-phosphono-pentanoic acid
-
irreversible inhibition
(S)-2-amino-5-phosphono-pent-4-ynoic acid
-
-
2'-phosphoribose AMP
-
-
2-Aminoadipate
3-Chloroacetylpyridine-adenine dinucleotide phosphate
-
NADP+ and NADPH protect
3-hydroxyaspartic acid
-
-
4-nitro-N,N-diethylbenzimidazolinone
4-nitro-N,N-dimethylbenzimidazolinone
4-nitro-N-ethylphthalimide
4-nitro-N-methylphthalimide
5-(carboxylatocarbonyl)-1H-pyrrole-2-carboxylate
5-[[(4-nitrophenyl)amino]carbonyl]-1,3-benzenedimethylcarboxylate
acetylenic and z-olefinic analogues
-
competitive inhibition
-
Adenosine 5'-triphosphate
-
causes a time-dependent inactivation at a concentration of 3.5 mM, 0C, pH 6.5 and 2 mM dithiothreitol, inactivation can be completely reversed by warming the reaction mixture to 25C, 50% inactivation occurs at a concentration of 2.5 mM, NADH protects
aromatic aldehydes
-
e.g.: benzaldehyde, weak
cis-5-phosphonic acid pipecolic acid
-
-
cysteine
-
in the reverse reaction
D-Cystine
-
70% inhibition at 0.01 mM, binds via the cysteine moiety covalently to the catalytic Cys135 of the enzyme, pH-dependent proces, optimal at pH 7.0-7.5, inhibition is reversible by DTT, dithioerythritol, 2-mercaptoethanol, dimercaptopropanol, and reduced glutathione, no protection by aspartate-beta-semialdehyde, NADP+ or NADPH, inhibition mechanism and kinetics
dimethyl pyridine-2,5-dicarboxylate
DTNB
-
reversible by DTT, dithioerythritol, 2-mercaptoethanol, dimercaptopropanol, and reduced glutathione
formaldehyde
-
-
Glutaraldehyde
-
-
GSSG
-
oxidized glutathione
homocysteine
-
in the reverse reaction
iodoacetamide
-
at 0.1 mM: 45.4% inactivation in the absence of NADP+, 22% inactivation in the presence of 1 mM NADP+
iodoacetate
L-2-Amino-4-oxo-5-chloropentanoic acid
L-cystine
L-cystine diethyl ester
-
68% inhibition at 0.01 mM, reversible by DTT, dithioerythritol, 2-mercaptoethanol, dimercaptopropanol, and reduced glutathione
L-cystine dimethyl ester
-
67% inhibition at 0.01 mM, reversible by DTT, dithioerythritol, 2-mercaptoethanol, dimercaptopropanol, and reduced glutathione
L-cystine hydroxamate
-
20% inhibition at 0.01 mM, reversible by DTT, dithioerythritol, 2-mercaptoethanol, dimercaptopropanol, and reduced glutathione
L-isoleucine
L-leucine
-
inhibits, when added to a final concentration of 10 mM in the assay system produces a decrease of 0.004 units in specific activity
L-lysine
L-methionine
L-threonine
methyl 5-nitropyridine-2-carboxylate
N-ethylmaleimide
NADPH-Tris-chloride buffer
-
promotes a weak inactivation at 0C, NADH protects
p-hydroxymercuribenzoate
-
-
Periodate
-
-
perrhenate
-
very weak inhibitor
-
petrosamine B
phosphonate
-
weak inhibitor
pipecolic acid-5-(R)-phosphate hydrochloric acid
-
-
pipecolic acid-5-(S)-phosphate hydrochloric acid
-
-
potassium phosphate
-
at a concentration of 10 mM promotes 60% inactivation at 0C in the presence of 10 mM ATP, at a concentration of 100 mM promotes 61% inactivation in the presence of 10 mM ATP and 32% inactivation in the absence of ATP, NADH protects
S-methyl cysteine sulfoxide
-
inhibitor binding structure deduced from crystal structure
S-methyl-L-cysteine sulfoxide
covalently binding inhibitor via Cys134 at the active site, inactivation, inhibition and binding mechanism, reversible by addition of DTT or 2-mercaptoethanol
tellurate
-
-
thieno[2,3-b]thiophene-2,5-dicarboxylate
trans-5-phosphonic acid pipecolic acid
-
-
Tris salts
tungstate
-
-
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
H2CO3
-
enhances activity 4 or 5 times in both directions
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.6
arsenate
-
-
140
cacodylate
-
recombinant wild-type enzyme, pH 9.0, 30C
6
HAsO42-
-
-
0.16
L-4-aspartyl phosphate
0.12 - 2.6
L-aspartate 4-semialdehyde
0.03 - 0.955
L-aspartate-4-semialdehyde
0.013 - 1.6
NADP+
0.02 - 3.3
NADPH
0.032 - 240
phosphate
0.14
vanadate
-
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
15.6
aspartyl phosphate
Arabidopsis thaliana
Q9FVC4
-
0.12 - 330
L-aspartate 4-semialdehyde
0.24 - 330
L-aspartate-4-semialdehyde
2
L-aspartate-semialdehyde
Streptococcus pneumoniae
-
-
3.2 - 330
NADP+
330
phosphate
Haemophilus influenzae
-
recombinant wild-type enzyme, pH 9.0, 30C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
236
L-aspartate 4-semialdehyde
Salmonella enterica
-
in 0.2 M bicine buffer, pH 8.6, at 25C
573
265
phosphate
Salmonella enterica
-
in 0.2 M bicine buffer, pH 8.6, at 25C
16
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.11 - 0.18
(1R)-5-[(4-nitrophenyl)carbamoyl]cyclohexa-3,5-diene-1,3-dicarboxylate
0.69 - 1.2
(2R,5R)-2,3,4,5-tetrahydropyridine-2,5-dicarboxylate
1.2 - 2.1
(2R,5R)-5-nitro-2,3,4,5-tetrahydropyridine-2-carboxylate
0.4 - 1.1
(3aR)-5-nitro-1,3,3a,4-tetrahydro-2H-benzimidazol-2-one
2.6 - 3.3
(3aR)-5-nitro-3a,4-dihydro-1H-isoindole-1,3(2H)-dione
0.15 - 0.18
(3aR)-6-chloro-5-nitro-3a,4-dihydro-1H-isoindole-1,3(2H)-dione
1.2
(S)-2-amino-5-fluoro-4-oxo-5-phosphono-pentanoic acid
-
0.2 M Tris, 1 mM EDTA, pH 8.6, 15 mM phosphate, 0.15 mM NADP+, 37C
3.9
(S)-2-amino-5-phosphono-pent-4-ynoic acid
-
0.2 M Tris, 1 mM EDTA, pH 8.6, 15 mM phosphate, 0.15 mM NADP+, 37C
0.05
2'-phosphoribose AMP
-
-
0.04
3-Chloroacetylpyridine-adenine dinucleotide phosphate
-
competitive inhibitor with respect to NADP+
4
4-nitro-N,N-diethylbenzimidazolinone
0.086
4-nitro-N,N-dimethylbenzimidazolinone
4
4-nitro-N-ethylphthalimide
0.89 - 1.1
4-nitro-N-methylphthalimide
20
5-[[(4-nitrophenyl)amino]carbonyl]-1,3-benzenedimethylcarboxylate
20
dimethyl pyridine-2,5-dicarboxylate
10
homocysteine
-
-
20
methyl 5-nitropyridine-2-carboxylate
0.22
Periodate
-
-
140
perrhenate
-
-
-
17
phosphonate
-
-
11
tellurate
-
-
26
tungstate
-
-
20
z-olefin
-
-
-
additional information
additional information
-
inhibition kinetics at 21C and pH 7.5
-
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.306
petrosamine B
Helicobacter pylori
-
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.002
-
K12 6201 strain, enriched media plus diaminopimelic acid
0.0029
-
of the recombinant in the crude extracts of Escherichia coli
0.01
-
grown in minimal medium or in minimal medium plus lysine, methionine and threonine
0.012
-
grown in minimal medium plus isoleucine
0.013
-
grown in minimal medium plus lysine or in minimal medium plus threonine
0.015
-
grown in minimal medium plus leucine or in minimal medium plus methionine
0.02
-
grown in complex medium
0.04
-
assay system plus lysine or assays system plus lysine, threonine and methionine
0.043
-
no addition to the assay system or assay system plus methionine
0.048
-
assay system plus threonine
0.12
-
B AC70R1 strain, enriched media
0.215
-
gene asd1
0.373
partially purified recombinant enzyme
0.4
-
K12 23631 strain, minimal media plus N-formyllysine
0.94
-
B AC70R1 strain, minimal media
1.8
-
K12 6201/PoP126 strain, enriched media plus ampicillin
2.149
-
gene asd2; when expressed in Escherichia coli
2.7
-
B AC70R1/PoP12 strain, enriched media plus tetracycline
5.3
-
B AC70R1/PoP12 strain, minimal media plus tetracycline
145
-
K12 23631/pOP12, after three times purification of the enzyme
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.4 - 7.8
-
mutant
7.5
-
assay at
8 - 9
-
L-aspartate 4-semialdehyde + arsenate
8
-
L-aspartyl phosphate + NADPH
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 9
-
pH 6.5: about 45% of activity maximum, mutant, about 8% of activity maximum, wild type, pH 9.0: about 55% of activity maximum, mutant, about 30% of activity maximum, wild type
8.4 - 9.5
-
at pH 8.4 and pH 9.5: 50% of maximal activity, L-aspartate 4-semialdehyde + phosphate
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
-
activity assay performed at room temperature
23
-
activity assay
25
-
assay at
28
-
above 28C, wild type
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Burkholderia thailandensis (strain E264 / ATCC 700388 / DSM 13276 / CIP 106301)
Candida albicans (strain SC5314 / ATCC MYA-2876)
Cryptococcus neoformans var. neoformans serotype D (strain JEC21 / ATCC MYA-565)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Filobasidiella neoformans
Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)
Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)
Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)
Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)
Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)
Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)
Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)
Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)
Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)
Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)
Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)
Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)
Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Streptococcus pneumoniae (strain ATCC BAA-255 / R6)
Streptococcus pneumoniae (strain ATCC BAA-255 / R6)
Streptococcus pneumoniae serotype 4 (strain ATCC BAA-334 / TIGR4)
Streptococcus pneumoniae serotype 4 (strain ATCC BAA-334 / TIGR4)
Streptococcus pneumoniae serotype 4 (strain ATCC BAA-334 / TIGR4)
Streptococcus pneumoniae serotype 4 (strain ATCC BAA-334 / TIGR4)
Sulfolobus tokodaii (strain DSM 16993 / JCM 10545 / NBRC 100140 / 7)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
38070
-
the expressed and purified monomer protein construct includes the 17-amino-acid sequence RGSHHHHHHGSACELGT between the N-terminal Met and the second amino acid Gly of the native Asd sequence, the total length of this protein is 362 amino acids
70000
-
gel filtration
75000
-
homodimer in solution, determined by gel filtration; recombinant His-tagged enzyme, gel filtration
77000
-
sucrose density gradient centrifugation
77500
-
ultracentrifugation
140000
-
gel filtration
156000
-
sedimentation equilibrium, meniscus depletion technique
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
tetramer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion method, using 20% (w/v) PEG 400, 0.1 M HEPES pH 6.5, 0.1 M MgCl2, at 20C
enzyme in open and closed form, 30 mg/ml purified recombinant enzyme in 10 mM Tris, pH 7.4, 40 mM KCl, with equal volume of reservoir solution, 0.006 ml sitting drops by vapour diffusion utilizing micro-bridges, 20% v/v ethylene glycol, 4C, X-ray diffraction structure determination and analysis at 1.95 A and 1.6 A resolution, respectively, modeling
-
using the hanging drop method
-
10 mg/ml purified recombinant wild-type and mutant enzymes free or in complex with the substrates, protein in 10 mM HEPES, pH 7.0, 1 mM EDTA, 1 mM DTT, by hanging drop vapour diffusion, 20C, mixed with equal volume of precipitant solution containing 22-24% PEG 3350, and 0.2 M ammonium acetate, crystals are soaked for 1 h in a solution containing 2 mM aspartate-beta-semialdehyde or 100 mM phosphate, 26% PEG 3350, 0.2 M ammonium acetate, 0.1 M Tris-HCl, pH 8.5, and 20% glycerol, X-ray diffraction structure determination and analysis at about 2.0 A resolution
-
15 mg/ml purified recombinant enzyme, in 10 mM HEPES, pH 7.0, 1 mM EDTA, 1 mM DTT, crystallized as apoenzyme, as hemithioacetal, or as hemithioacetal structure with bound phosphate, hanging drop vapour diffusion method, 20C, 1:1 mixture of protein solution and precipitant solution, the latter containing 24-28% PEG 3350, 0.2 M ammonium acetate, 0.1 M Tris, pH 8.5, overnight, substrate complexing by soaking of crystals in mother liquor with 50 mM potassium phosphate, crystals are frozen in precipitant solution with 20% glycerol added, X-ray diffraction structure determination and analysis at 2.0-2.15 A resolution, modeling
-
15 mg/ml purified recombinant wild-type enzyme in 10 mM HEPES, pH 7.0, 1 mM EDTA, and 1 mM DTT, enzyme is free or complexed with substrates phosphate and/or asparate-beta-semialdehyde, hanging drop vapour diffusion method, 20C, against an equal volume of precipitant solution containing 24-28% PEG 3350, 0.2 M ammonium acetate, and 0.1 M Tris-HCl, pH 8.5, soaking of crystals before harvest in 100 mM phosphate and 2 mM aspartate-beta-semialdehyde, crystallization of mutant H277N in 10 mM HEPES, pH 7.0, 1 mM EDTA, and 1 mM DTT, by addition of precipitant solution containing 5 mM NADP+ and 5 mM inhibitor S-methyl-L-cysteine sulfoxide, 22% PEG 3350, 0.2 M ammonium acetate and 0.1 M sodium cacodylate, pH 6.5, X-ray diffraction structure determination and analysis at about 2.0 A resolution
-
about 10 mg/ml pure recombinant wild-type enzyme in 10 mM HEPES, pH 7.0, 1 mM EDTA, and 1 mM DTT, complexed with oxyanions arsenate or periodate, hanging drop vapour diffusion method, 20C, against an equal volume of precipitant solution containing 22-24% PEG 4000, 0.2 M ammonium acetate, and Tris-HCl, pH 8.5, soaking of crystals before harvest in a solution containing 26% PEG3350, 0.2 M ammonium acetate, 100 mM periodate or arsenate,0.1 M Tris-HCl, pH 8.5, and 20% glycerol, X-ray diffraction structure determination and analysis at 2.3 A resolution
-
2.3 A resolution; the structure of aspartate-beta-semialdehyde dehydrogenase has been determined to 2.3 A resolution using multiwavelength anomalous diffraction phasing of a seleno-methionine-substituted derivative
-
by using the hanging-drop vapour-diffusion method, crystallization in 2 different crystal forms, diffraction data analysis suggests the presence of up to four monomers in the asymmetric unit of the orthorhombic crystal form and of one or 2 monomers in the cubic crystal form; purified recombinant His-tagged enzyme, 9 mg/ml protein in 10 mM potassium phosphate buffer, pH 8.0, and 10 mM DTT, sitting drop vapour diffusion method, mixing of 500 nl of protein and of reservoir solution, the latter containing 1.6 M ammonium sulfate and 100 mM citric acid, pH 5.0, 2 days at 18C, two different crystal forms, X-ray diffraction structure determination and analysis at 2.18-2.75 A resolution
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in complex with S-methyl-l-cysteine sulfoxide and sulfate, sitting drop vapor diffusion method, using 1.6 M ammonium sulfate and 100 mM citric acid pH 5.0
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; crystallization of the apo-enzyme, in complex with NADP+, in complex with L-aspartate-beta-semialdehyde, in complex with NADP+ and L-aspartate-beta-semialdehyde
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12 mg/ml purified recombinant enzyme free or in ternary complex with NADP+ and covalently bound inhibitor S-methyl-L-cysteine sulfoxide, protein in 10 mM HEPES, pH 7.0, 1 mM EDTA, and 1 mM DTT, hanging drop vapour diffusion method, 20C, with or without 5 mM NADP+ and 5 mM inhibitor, against an equal volume of precipitant solution: containing 18% PEG 3350, 0.2 M sodium acetate, and 0.1 M Tris, pH 8.5 for the free enzyme, or containing 22% PEG 3350, 0.2 M sodium acetate, and 0.1 M sodium citrate, pH 5.6 for the ternary complex, addition of 20% glycerol for crystal freezing, X-ray diffraction structure determination and analysis at 2.8 A and 1.84 A resolution, respectively
apoenzyme and enzyme in complex with substrate L-aspartate semialdehyde, method optimization, purified protein in 50 mM sodium citrate, pH 5.6, with 0.2 M ammonium acetate and 2 mM DTT via dialysis overnight, hanging drop vapour diffusion method, 4C, diluted back into 100 mM Tris, pH 8.5, with 200 mM ammonium acetate and 5 mM DTT with 12 mg/ml protein, 0.001 ml protein solution is mixed with 0.001 ml of precipitant containing 0.1 M sodium citrate, pH 5.5-6.5, 5 mM DTT, 0.1-0.4 M ammonium acetate, and 24-27% PEG 8000, method optimization, overview, X-ray diffraction structure determination and analysis at 2.0-2.2 A resolution, molecular replacement method
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
65
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30 min, wild type, stable
additional information
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stability of recombinant wild-type and mutant enzymes, oveview
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
adenosine 5'-triphosphate, time dependent inactivation at 0C
-
lyophilization of purified or partially purified enzyme, slight loss of activity
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 10 mM potassium phosphate, pH 7.2, 0.1 mM EDTA, 1 mM dithioerythritol, 50% glycerol, 2 months
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-80C, no noticeable loss of activity
4C, 10 mM potassium phosphate, pH 7.2, 0.1 mM EDTA, 1 mM dithioerythritol, several days
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frozen or on ice, after dialysis, 1 month stable
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
anion exchange chromatography and gel-filtration; using anion-exchange chromatography, Source 15Q, followed by gel filtration
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by using affinity chromatography and gel filtration; recombinant His-tagged enzyme from Escherichia coli strain M15 by nickel affinity chromatography and gel filtration
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cobalt immobilized metal-affinity column chromatography and Source 30Q column chromatography
K-12, using ammonium sulfate precipitation and column chromatography on Sephadex G-200 and DEAE-Sephadex A-50
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Ni-affinity chromatography
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Ni-affinity chromatography and anion exchange chromatography; using a Co2+ affinity matrix and anion exchange chromatography
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Ni-NTA column chromatography and gel filtration
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partially, recombinant enzyme from Escherichia coli, 37fold
recombinant enzyme from Escherichia coli to over 95% purity by anion exchange chromatography and gel filtration
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recombinant enzyme from strain BL21(DE3) in several steps
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recombinant His-tagged enzyme from Escherichia coli by nickel affinity chromatography to 96% purity
to homogeneity
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using ammonium sulfate precipitation and column chromatography on Q Sepharose XL and Procion Red-A
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using ammonium sulfate precipitation and column chromatography on Sephadex G-200, DEAE-cellulose and hydroxylapatite
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using anionic exchange chromatography on DEAE EMD 650 column and Q-Sepharose column
using column chromatography on DEAE-cellulose, DEAE-Sephadex A-50, hydroxylapatite, ultrogel ACA-44 and Sepharose C3
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using column chromatography on Q Sepharose XL and omega-aminohexyl-agarose for the purification of V. cholerae I and column chromatography on Q Sepharose XL for the purification of V. cholerae II
using column chromatography on Q Sepharose XL and phenyl-Sepharose
using heat treatment, acid precipitation, protamine sulfate treatment and ammonium sulfate precipitation
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using heat treatment, column chromatography on DEAE-cellulose, ammonium sulfate precipitation and column chromatography on Sephadex G-200
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using protamine sulfate treatment, ammonium sulfate precipitation, chromatography on DEAE-Sephadex A-50 column, Bio-Gel hydroxylapatite phosphate column, Bio-Gel hydroxylapatite sulfate column, acid treatment and column chromatography on Sephadex G-200
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning and recombinant protein overproduction with Escherichia coli
co-expression with L-lysine-insensitive aspartokinase, dihydrodipicolinate synthase, and dihydrodipicolinate reductase in the parent strain and in an AEC-resistant mutant strain of Lactobacillus plantarum from a shuttle vector, leading to increased L-lysine production, but absolutely requiring overexpression of aspartokinase or dihydrodipicolinate synthase, overview
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expressed as inclusion bodies with an N-terminal hexahistidine tag in pET28b vector or subcloned into pET41 vector with NdeI/SalI
expressed in Escherichia coli
expressed in Escherichia coli BL21 cells
expressed in Escherichia coli M15 cells
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expressed in Escherichia coli, the genome contains two genes for the enzyme
expression as His-tag fusion protein in Escherichia coli BL21 (DE3); into a pET28a vector for expression in Escherichia coli BL21DE3
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expression Escherichia coli
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expression in Escherichia coli
expression in Escherichia coli Hfr3000; genes are amplified from chromosomal DNA of Lactobacillus plantarum and cloned into the pCR2.1-TOPO vector for sequencing, and subcloned into the pUC18 vector for heterologous expression in Escherichia coli
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expression in Escherichia coli; into the pET41-41a vector for transformation into Rosetta-competent Escherichia coli cells
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expression of His-tagged enzyme in Escherichia coli strain M15; heterologous expression in Escherichia coli
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expression of wild-type and mutant enzymes
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gene asd, cloning from genomic DNA library, DNA sequence determination and analysis, expression in enzyme-deficient auxotrophic Escherichia coli strain CGSC5080
gene asd, subcloning in Escherichia coli strain JM109, DNA sequence determination and analysis, optimization of functional overexpression in Escherichia coli strain M15 as His-tagged protein
HOM2 gene encodes aspartic semialdehyde dehydrogenase
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recombinant large scale expression in strain BL21(DE3)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
R267L
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the mutant shows 9.5% activity compared to the wild type enzyme
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
drug development
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ASADH is a target for the development of novel antibiotics, especially for Gram-negative bacteria that require diaminopimelate for cell-wall biosynthesis
medicine
pharmacology
additional information
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