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Literature summary for 1.4.3.3 extracted from

  • Mueller, M.; Kratzer, R.; Schiller, M.; Slavica, A.; Rechberger, G.; Kollroser, M.; Nidetzky, B.
    The role of Cys108 in Trigonopsis variabilis D-amino acid oxidase examined through chemical oxidation studies and point mutations C108S and C108D (2010), Biochim. Biophys. Acta, 1804, 1483-1491.
    View publication on PubMed
Search for more literature for 1.4.3.3

Cloned(Commentary)

Cloned (Comment) Organism
expression of Strep-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 (DE3), induction by DL-Ala Trigonopsis variabilis

Protein Variants

Protein Variants Comment Organism
C108D site-directed mutagenesis, in contrast to the wild-type enzyme, the mutant releases the cofactor in a quasi-irreversible manner and is therefore not stabilized by external FAD against loss of activity. Conformational properties and kinetics of C108D, overview Trigonopsis variabilis
C108S site-directed mutagenesis, in contrast to the wild-type enzyme, the mutant releases the cofactor in a quasi-irreversible manner and is therefore not stabilized by external FAD against loss of activity. Conformational properties and kinetics of C108S, overview Trigonopsis variabilis
R110A site-directed mutagenesis, the mutant shows activity similar to the wild-type enzyme, but shows increased temperature denaturation. Release of FAD is the dominant path of thermal denaturation of R110A Trigonopsis variabilis

Inhibitors

Inhibitors Comment Organism Structure
additional information oxidative modification of Trigonopsis variabilis D-amino acid oxidase in vivo is traceable as the conversion of Cys108 into a stable cysteine sulfinic acid, causes substantial loss of activity and thermostability of the enzyme Trigonopsis variabilis

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
D-methionine + H2O + O2 Trigonopsis variabilis
-
 4-methylsulfanyl-2-oxobutanoate + NH3 + H2O2
-
 ?
D-methionine + H2O + O2 Trigonopsis variabilis ATCC 10679
-
 4-methylsulfanyl-2-oxobutanoate + NH3 + H2O2
-
 ?

Organism

Organism UniProt Comment Textmining
Trigonopsis variabilis
-
-
-
Trigonopsis variabilis ATCC 10679
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant Strep-tagged wild-type and mutant enzymes from Escherichia coli strain BL21 (DE3) by ultracentrifugation, affinity and anion exchange chromatography Trigonopsis variabilis

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
2.2
-
 recombinant Strep-tagged wild-type enzyme, substrates are D-methionine and 6-dichloroindophenolate, pH 7.5, 37°C Trigonopsis variabilis
2.6
-
 recombinant Strep-tagged mutant R101A, substrates are D-methionine and 6-dichloroindophenolate, pH 7.5, 37°C Trigonopsis variabilis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
D-methionine + 2,6-dichloroindophenol
-
 Trigonopsis variabilis 4-methylsulfanyl-2-oxobutanoate + reduced 2,6-dichloroindophenol
-
 ?
D-methionine + 2,6-dichloroindophenol
-
 Trigonopsis variabilis ATCC 10679 4-methylsulfanyl-2-oxobutanoate + reduced 2,6-dichloroindophenol
-
 ?
D-methionine + H2O + O2
-
 Trigonopsis variabilis 4-methylsulfanyl-2-oxobutanoate + NH3 + H2O2
-
 ?
D-methionine + H2O + O2 with 6-dichloroindophenol as electron acceptor Trigonopsis variabilis 4-methylsulfanyl-2-oxobutanoate + NH3 + H2O2
-
 ?
D-methionine + H2O + O2
-
 Trigonopsis variabilis ATCC 10679 4-methylsulfanyl-2-oxobutanoate + NH3 + H2O2
-
 ?
D-methionine + H2O + O2 with 6-dichloroindophenol as electron acceptor Trigonopsis variabilis ATCC 10679 4-methylsulfanyl-2-oxobutanoate + NH3 + H2O2
-
 ?

Subunits

Subunits Comment Organism
homodimer
-
 Trigonopsis variabilis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Trigonopsis variabilis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.84
-
 D-methionine recombinant mutant C108S, pH 7.5, 37°C, with 6-dichloroindophenolate Trigonopsis variabilis
1.5
-
 D-methionine recombinant wild-type enzyme, pH 7.5, 37°C, with 6-dichloroindophenolate Trigonopsis variabilis
2.8
-
 D-methionine recombinant mutant C108D, pH 7.5, 37°C, with 6-dichloroindophenolate Trigonopsis variabilis
75
-
 D-methionine recombinant mutant C108D, pH 7.5, 37°C, with O2 Trigonopsis variabilis
110
-
 D-methionine recombinant mutant C108S, pH 7.5, 37°C, with O2 Trigonopsis variabilis
200
-
 D-methionine recombinant wild-type enzyme, pH 7.5, 37°C, with O2 Trigonopsis variabilis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
 assay at Trigonopsis variabilis

Cofactor

Cofactor Comment Organism Structure
FAD
-
 Trigonopsis variabilis

Expression

Organism Comment Expression
Trigonopsis variabilis DL-Ala induces the enzyme up

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
153
-
 D-methionine recombinant mutant C108S, pH 7.5, 37°C Trigonopsis variabilis
258
-
 D-methionine recombinant mutant C108D, pH 7.5, 37°C Trigonopsis variabilis
274
-
 D-methionine recombinant wild-type enzyme, pH 7.5, 37°C Trigonopsis variabilis