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Literature summary for 1.4.3.3 extracted from
- A single Phe54Tyr substitution improves the catalytic activity and thermostability of Trigonopsis variabilis D-amino acid oxidase (2010), New Biotechnol., 27, 78-84.
Application
| Application | Comment | Organism |
|---|---|---|
| synthesis | faster substrate turnover, reduced glutaryl-7-aminocephalosporanic acid inhibition and improved thermostability of the F54Y mutant compared to the wild-type enzyme render it a useful candidate in industrial production of semi-synthetic cephems | Trigonopsis variabilis |
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| expression of wild-type enzyme and mutants F54Y in Escherichia coli BL21(DE3) | Trigonopsis variabilis |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| F54A | site-directed mutagenesis | Trigonopsis variabilis |
| F54S | site-directed mutagenesis | Trigonopsis variabilis |
| F54Y | site-directed mutagenesis, the mutant shows 6fold improvement in kcat,app and about 2.5fold increase in Ki of glutaryl-7-aminocephalosporanic acid, the substitution improves the catalytic activity and thermostability of mutant DAAO compared to the wild-type enzyme. Heat treatment at 55° for 60 min does not decrease the activity of F54Y. The Tyr substitution might initiate hydrogen bond formation with the amino group of CPC and facilitate deamination | Trigonopsis variabilis |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| glutaryl-7-aminocephalosporanic acid | - |
Trigonopsis variabilis |  |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.9 | - |
cephalosporin C | recombinant mutant F54S, pH 7.5, 22°C | Trigonopsis variabilis | |
| 1.2 | - |
cephalosporin C | recombinant mutant F54A, pH 7.5, 22°C | Trigonopsis variabilis | |
| 1.6 | - |
cephalosporin C | recombinant wild-type enzyme, pH 7.5, 22°C | Trigonopsis variabilis | |
| 4.8 | - |
cephalosporin C | recombinant mutant F54Y, pH 7.5, 22°C | Trigonopsis variabilis | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Trigonopsis variabilis | Q6R4Q9 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant wild-type enzyme and mutants from Escherichia coli BL21(DE3) | Trigonopsis variabilis |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 8.5 | - |
purified recombinant wild-type enzyme, substrate cephalosporin C, pH 7.5, 22°C | Trigonopsis variabilis |
| 22.3 | - |
purified recombinant mutant F54S, substrate cephalosporin C, pH 7.5, 22°C | Trigonopsis variabilis |
| 26.4 | - |
purified recombinant mutant F54A, substrate cephalosporin C, pH 7.5, 22°C | Trigonopsis variabilis |
| 30.6 | - |
purified recombinant mutant F54Y, substrate cephalosporin C, pH 7.5, 22°C | Trigonopsis variabilis |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| cephalosporin C + H2O + O2 | conversion of cephalosporin C to 7-aminocephalosporanic acid and spontaneous decarboxylation of oxoadipyl-7-amino cephalosporanic acid is promoted by the H2O2 formed in the oxidase reaction of TvDAO | Trigonopsis variabilis | 7-aminocephalosporanic acid + ? + H2O2 | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| DAAO | - |
Trigonopsis variabilis |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 22 | - |
assay at | Trigonopsis variabilis |
Temperature Stability [°C]
| Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 50 | 65 | - |
Trigonopsis variabilis |
| 55 | - |
TvDAAO is active after heat treatment for 30 min | Trigonopsis variabilis |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 6.167 | - |
cephalosporin C | recombinant wild-type enzyme, pH 7.5, 22°C | Trigonopsis variabilis | |
| 16.67 | - |
cephalosporin C | recombinant mutant F54S, pH 7.5, 22°C | Trigonopsis variabilis | |
| 18.33 | - |
cephalosporin C | recombinant mutant F54A, pH 7.5, 22°C | Trigonopsis variabilis | |
| 36.67 | - |
cephalosporin C | recombinant mutant F54Y, pH 7.5, 22°C | Trigonopsis variabilis | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7.5 | - |
assay at | Trigonopsis variabilis |
pH Stability
| pH Stability | pH Stability Maximum | Comment | Organism |
|---|---|---|---|
| 6 | 10 | - |
Trigonopsis variabilis |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| FAD | - |
Trigonopsis variabilis | |
Ki Value [mM]
| Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.7 | - |
glutaryl-7-aminocephalosporanic acid | recombinant mutant F54A, pH 7.5, 22°C | Trigonopsis variabilis | |
| 1.1 | - |
glutaryl-7-aminocephalosporanic acid | recombinant mutant F54S, pH 7.5, 22°C | Trigonopsis variabilis | |
| 1.5 | - |
glutaryl-7-aminocephalosporanic acid | recombinant wild-type enzyme, pH 7.5, 22°C | Trigonopsis variabilis | |
| 3.8 | - |
glutaryl-7-aminocephalosporanic acid | recombinant mutant F54Y, pH 7.5, 22°C | Trigonopsis variabilis | |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 3.83 | - |
cephalosporin C | recombinant wild-type enzyme, pH 7.5, 22°C | Trigonopsis variabilis | |
| 7.83 | - |
cephalosporin C | recombinant mutant F54Y, pH 7.5, 22°C | Trigonopsis variabilis | |
| 15.67 | - |
cephalosporin C | recombinant mutant F54A, pH 7.5, 22°C | Trigonopsis variabilis | |
| 18.33 | - |
cephalosporin C | recombinant mutant F54S, pH 7.5, 22°C | Trigonopsis variabilis | |
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