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Literature summary for 1.4.3.1 extracted from

  • Takahashi, S.; Matsumoto, S.; Maruyama, K.; Wakaizumi, A.; Abe, K.; Kera, Y.; Yamada, R.
    An active-site mutation enhances the catalytic activity of the yeast Cryptococcus humicola D-aspartate oxidase (2009), J. Mol. Catal. B, 61, 235-240.
No PubMed abstract available
Search for more literature for 1.4.3.1

Cloned(Commentary)

Cloned (Comment) Organism
expression of His-tagged wild-type and mutant DDOs in Escherichia coli strain Rosetta (DE3) Vanrija humicola

Protein Variants

Protein Variants Comment Organism
R243A site-directed mutagenesis, inactive mutant Vanrija humicola
R243D site-directed mutagenesis, the mutant exhibits different spectral features of bound flavin from that of the wild-type enzyme. R243D exhibits approximately 2fold higher DDO activity than that of the wild-type with a high substrate specificity to D-aspartate. Kinetic analysis shows that R243D mutant has a 2.5fold lower Km and a 1.6fold higher kcat for D-aspartate compared to the wild-type enzyme, resulting in approximately 4fold higher catalytic efficiency Vanrija humicola
R243E site-directed mutagenesis, the mutant exhibits different spectral features of bound flavin from that of the wild-type enzyme and displays lower DDO activity. The mutant also oxidizes the substrates of DAO, D-Ala and D-Arg, albeit with very low activities Vanrija humicola
R243K site-directed mutagenesis, the mutant exhibits different spectral features of bound flavin from that of the wild-type enzyme and displays lower DDO activity Vanrija humicola
R243M site-directed mutagenesis, the mutant exhibits different spectral features of bound flavin from that of the wild-type enzyme and displays lower DDO activity. The mutant also oxidizes the substrates of DAO, D-Ala and D-Arg, albeit with very low activities Vanrija humicola

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information kinetics of wild-type and mutant enzymes, overview Vanrija humicola
1
-
 D-Asp pH 7.0, 20°C, recombinant mutant R243D Vanrija humicola
2
-
 D-Asp pH 7.0, 20°C, recombinant mutant R243E Vanrija humicola
2.5
-
 D-Asp pH 7.0, 20°C, recombinant wild-type enzyme Vanrija humicola
2.6
-
 D-Glu pH 7.0, 20°C, recombinant wild-type enzyme Vanrija humicola
3.8
-
 D-Asp pH 7.0, 20°C, recombinant mutant R243M Vanrija humicola
10.3
-
 D-Asp pH 7.0, 20°C, recombinant mutant R243K Vanrija humicola
13.4
-
 D-Asp pH 7.0, 20°C, recombinant mutant R243A Vanrija humicola
15.7
-
 N-methyl-D-Asp pH 7.0, 20°C, recombinant wild-type enzyme Vanrija humicola
27.9
-
 N-methyl-D-Asp pH 7.0, 20°C, recombinant wild-type enzyme Vanrija humicola
106
-
 D-Glu pH 7.0, 20°C, recombinant wild-type enzyme Vanrija humicola

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
D-Asp + H2O + O2 Vanrija humicola
-
 oxaloacetate + NH3 + H2O2
-
 ?

Organism

Organism UniProt Comment Textmining
Vanrija humicola Q75WF1 i.e. Asterotremella humicola
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant DDOs from Escherichia coli strain Rosetta (DE3) by metal affinity chromatography and gel filtration Vanrija humicola

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
 mutant activities with D-Ala and D-Arg, overview Vanrija humicola
0.04
-
 pH 7.0, 20°C, purified recombinant mutant R243E, substrate N-methyl-D-Asp Vanrija humicola
0.04
-
 pH 7.0, 20°C, purified recombinant mutant R243K, substrate N-methyl-D-Asp Vanrija humicola
0.09
-
 pH 7.0, 20°C, purified recombinant mutant R243A, substrate D-Asp Vanrija humicola
0.5
-
 pH 7.0, 20°C, purified recombinant mutant R243K, substrate D-Glu Vanrija humicola
1
-
 pH 7.0, 20°C, purified recombinant mutant R243E, substrate D-Glu Vanrija humicola
1.2
-
 pH 7.0, 20°C, purified recombinant mutant R243D, substrate N-methyl-D-Asp Vanrija humicola
1.5
-
 pH 7.0, 20°C, purified recombinant mutant R243K, substrate D-Asp Vanrija humicola
1.6
-
 pH 7.0, 20°C, purified recombinant wild-type enzyme, substrate D-Glu Vanrija humicola
1.9
-
 pH 7.0, 20°C, purified recombinant mutant R243E, substrate D-Asp Vanrija humicola
9.2
-
 pH 7.0, 20°C, purified recombinant wild-type enzyme, substrate N-methyl-D-Asp Vanrija humicola
13.1
-
 pH 7.0, 20°C, purified recombinant mutant R243M, substrate D-Glu Vanrija humicola
18.5
-
 pH 7.0, 20°C, purified recombinant mutant R243D, substrate D-Glu Vanrija humicola
86
-
 pH 7.0, 20°C, purified recombinant mutant R243M, substrate D-Asp Vanrija humicola
93.7
-
 pH 7.0, 20°C, purified recombinant wild-type enzyme, substrate D-Asp Vanrija humicola
181
-
 pH 7.0, 20°C, purified recombinant mutant R243D, substrate D-Asp Vanrija humicola

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
D-Asp + H2O + O2
-
 Vanrija humicola oxaloacetate + NH3 + H2O2
-
 ?
D-Asp + H2O + O2 arginine residue 243 is possibly involved in the substrate recognition, active site model, overview Vanrija humicola oxaloacetate + NH3 + H2O2
-
 ?
D-Glu + H2O + O2
-
 Vanrija humicola 2-oxoglutarate + NH3 + H2O2
-
 ?
additional information substrate specificities of wild-type and mutants DDOs, overview Vanrija humicola ?
-
 ?
N-methyl-D-Asp + H2O + O2
-
 Vanrija humicola oxaloacetate + methylamine + H2O2
-
 ?

Subunits

Subunits Comment Organism
More three-dimensional model of ChDDO, comparison with the crystal structure of a yeast Rhodotorula gracilis D-amino acid oxidase, EC 1.4.3.3 Vanrija humicola

Synonyms

Synonyms Comment Organism
DDO
-
 Vanrija humicola

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
20
-
 assay at Vanrija humicola

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.3
-
 D-Asp pH 7.0, 20°C, recombinant mutant R243E Vanrija humicola
1.4
-
 D-Asp pH 7.0, 20°C, recombinant mutant R243K Vanrija humicola
62.6
-
 D-Asp pH 7.0, 20°C, recombinant mutant R243M Vanrija humicola
74.5
-
 D-Asp pH 7.0, 20°C, recombinant wild-type enzyme Vanrija humicola
121
-
 D-Asp pH 7.0, 20°C, recombinant mutant R243D Vanrija humicola

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
 assay at Vanrija humicola

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
24
-
 D-Glu pH 7.0, 20°C, recombinant wild-type enzyme Vanrija humicola
136
-
 D-Asp pH 7.0, 20°C, recombinant mutant R243K Vanrija humicola
562
-
 N-methyl-D-Asp pH 7.0, 20°C, recombinant wild-type enzyme Vanrija humicola
650
-
 D-Asp pH 7.0, 20°C, recombinant mutant R243E Vanrija humicola
16500
-
 D-Asp pH 7.0, 20°C, recombinant mutant R243M Vanrija humicola
29800
-
 D-Asp pH 7.0, 20°C, recombinant wild-type enzyme Vanrija humicola
121000
-
 D-Asp pH 7.0, 20°C, recombinant mutant R243D Vanrija humicola