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Literature summary for 5.1.1.13 extracted from

  • Washio, T.; Kato, S.; Oikawa, T.
    Molecular cloning and enzymological characterization of pyridoxal 5'-phosphate independent aspartate racemase from hyperthermophilic archaeon Thermococcus litoralis DSM 5473 (2016), Extremophiles, 20, 711-721 .
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
gene OCC_11152, sequence comparisons, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain Rosetta (DE3) Thermococcus litoralis

Protein Variants

Protein Variants Comment Organism
C194A site-directed mutagenesis, inactive mutant Thermococcus litoralis
C83A site-directed mutagenesis, inactive mutant Thermococcus litoralis
C83A/C194A site-directed mutagenesis, inactive mutant Thermococcus litoralis

Inhibitors

Inhibitors Comment Organism Structure
5,5'-dithiobis(2-nitrobenzoic acid) 35% inhibition at 1 mM Thermococcus litoralis
CoSO4 36% inhibition at 1 mM Thermococcus litoralis
CuSO4 15% inhibition at 1 mM Thermococcus litoralis
EDTA 7% inhibition at 1 mM Thermococcus litoralis
FeSO4 43% inhibition at 1 mM Thermococcus litoralis
iodoacetic acid complete inhibition at 1 mM Thermococcus litoralis
MgSO4 48% inhibition at 1 mM Thermococcus litoralis
MnSO4 18% inhibition at 1 mM Thermococcus litoralis
N-ethylmaleimide 62% inhibition at 1 mM Thermococcus litoralis
NiSO4 31% inhibition at 1 mM Thermococcus litoralis
p-chloromercuribenzoic acid 36% inhibition at 1 mM Thermococcus litoralis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information the activation energy of Tl-AspR is calculated to be 51.8 kJ/mol by Arrhenius plots Thermococcus litoralis
30.2
-
L-aspartate recombinant enzyme, pH 8.0, 90°C Thermococcus litoralis
136
-
D-Aspartate recombinant enzyme, pH 8.0, 90°C Thermococcus litoralis

Metals/Ions

Metals/Ions Comment Organism Structure
CaCl2 activates to 120% activity at 1 mM Thermococcus litoralis

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
56000
-
recombinant enzyme, gel filtration Thermococcus litoralis

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
L-aspartate Thermococcus litoralis
-
D-aspartate
-
r
L-aspartate Thermococcus litoralis ATCC 51850 / DSM 5473 / JCM 8560 / NS-C
-
D-aspartate
-
r

Organism

Organism UniProt Comment Textmining
Thermococcus litoralis H3ZNI2 isolated from a hot spring of a shallow sea in Naples, Italy
-
Thermococcus litoralis ATCC 51850 / DSM 5473 / JCM 8560 / NS-C H3ZNI2 isolated from a hot spring of a shallow sea in Naples, Italy
-

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type and mutant enzymes from Escherichia coli strain Rosetta (DE3) by heat treatment of cell-free extract at 70°C for 30 min, centrifugation, dialysis, and anion exchange chromatography, dialysis, hydrophobic interaction chromatography, and another different anion exchange chromatography, followed by dialysis Thermococcus litoralis

Source Tissue

Source Tissue Comment Organism Textmining
additional information the organism is able to grow at 55-98°C in pH 4.0-8.0, optimally at 88°C and pH 6.0 Thermococcus litoralis
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1550
-
crude recombinant, pH 8.0, 90°C Thermococcus litoralis
1590
-
purified recombinant enzyme after complete purification procedure, pH 8.0, 90°C Thermococcus litoralis
3870
-
partially purified recombinant enzyme, after the first anion exchange chromatographic step, pH 8.0, 90°C Thermococcus litoralis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
D-aspartate D-Asp is the preferred substrate Thermococcus litoralis L-aspartate
-
r
D-aspartate D-Asp is the preferred substrate Thermococcus litoralis ATCC 51850 / DSM 5473 / JCM 8560 / NS-C L-aspartate
-
r
L-aspartate
-
Thermococcus litoralis D-aspartate
-
r
L-aspartate 56.4% of the activity in the reverse reaction direction Thermococcus litoralis D-aspartate
-
r
L-aspartate
-
Thermococcus litoralis ATCC 51850 / DSM 5473 / JCM 8560 / NS-C D-aspartate
-
r
L-aspartate 56.4% of the activity in the reverse reaction direction Thermococcus litoralis ATCC 51850 / DSM 5473 / JCM 8560 / NS-C D-aspartate
-
r
additional information the enzyme also acts on L-cysteic acid and L-cysteine sulfinic acid (with below 30% activity compared to D-aspartate) in addition to D- and L-aspartic acids. No activity with D-glutamate, D-asparagine, D-glutamine, D-alanine, D-leucine, D-valine, D-proline, D-serine, D-threonine, and D-arginine Thermococcus litoralis ?
-
?
additional information the enzyme also acts on L-cysteic acid and L-cysteine sulfinic acid (with below 30% activity compared to D-aspartate) in addition to D- and L-aspartic acids. No activity with D-glutamate, D-asparagine, D-glutamine, D-alanine, D-leucine, D-valine, D-proline, D-serine, D-threonine, and D-arginine Thermococcus litoralis ATCC 51850 / DSM 5473 / JCM 8560 / NS-C ?
-
?

Subunits

Subunits Comment Organism
homodimer 2 * 25000-30000, recombinant enzyme, SDS-PAGE Thermococcus litoralis

Synonyms

Synonyms Comment Organism
OCC_11152
-
Thermococcus litoralis
PLP-independent AspR
-
Thermococcus litoralis
pyridoxal 5'-phosphate independent aspartate racemase
-
Thermococcus litoralis
Tl-AspR
-
Thermococcus litoralis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
95
-
the activity of Tl-AspR increases gradually and shows a maximum activity at 95°C Thermococcus litoralis

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
90
-
purified enzyme, approximately 50% of initial activity remains after incubation for 11 h Thermococcus litoralis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
118000
-
L-aspartate recombinant enzyme, pH 8.0, 90°C Thermococcus litoralis
610000
-
D-Aspartate recombinant enzyme, pH 8.0, 90°C Thermococcus litoralis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
-
Thermococcus litoralis

pH Stability

pH Stability pH Stability Maximum Comment Organism
6 8 purified enzyme, min. 50% activity remaining within this range Thermococcus litoralis

Cofactor

Cofactor Comment Organism Structure
additional information the enzyme does not require pyridoxal 5'-phosphate as a coenzyme Thermococcus litoralis

General Information

General Information Comment Organism
evolution the essential cysteine residues are conserved as Cys83 and Cys194 Thermococcus litoralis
additional information amino acid analysis of cell-free extract, overview Thermococcus litoralis

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3907.3
-
L-aspartate recombinant enzyme, pH 8.0, 90°C Thermococcus litoralis
4485.3
-
D-Aspartate recombinant enzyme, pH 8.0, 90°C Thermococcus litoralis