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Literature summary for 3.5.1.81 extracted from
- Increasing the storage and oxidation stabilities of N-acyl-D-amino acid amidohydrolase by site-directed mutagenesis of critical methionine residues (2012), Process Biochem., 47, 1785-1790.
No PubMed abstract available
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| recombinant expression of wild-type and mutant enzymes in Escherichia coli strain JM109 | Variovorax paradoxus |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| M221L | site-directed mutagenesis, the mutant shows 10% enhanced activity and a 44% decreased Km, but a 2.4fold increased kcat/Km compared to the wild-type enzyme, the mutant half-life at 4°C increases up to 6fold compared to the wild-type | Variovorax paradoxus |
| M254L | site-directed mutagenesis, the mutant shows 30% reduced activity, but increased half-life compared to the wild-type enzyme | Variovorax paradoxus |
| M352L | site-directed mutagenesis, the mutant shows 30% reduced activity, but similar half-life compared to the wild-type enzyme | Variovorax paradoxus |
| M39L | site-directed mutagenesis, the mutant shows 10% reduced activity, but increased half-life compared to the wild-type enzyme | Variovorax paradoxus |
| M56L | site-directed mutagenesis, the mutant shows 30% reduced activity, but similar half-life compared to the wild-type enzyme | Variovorax paradoxus |
General Stability
| General Stability | Organism |
|---|---|
| the recombinant enzyme is unstable during protein purification and storage at 4°C probably due to methionine oxidation | Variovorax paradoxus |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| additional information | Met221 spatial closeness to the zinc-assistant catalytic center is highly potential as the primary site for oxidative inactivation of the enzyme via its methionine residue | Variovorax paradoxus |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.0137 | - |
N-acetyl-D-methionine | recombinant mutant M254L, pH and temperature not specified in the publication | Variovorax paradoxus |  |
| 0.0144 | - |
N-acetyl-D-methionine | recombinant mutant M352L, pH and temperature not specified in the publication | Variovorax paradoxus | |
| 0.0155 | - |
N-acetyl-D-methionine | recombinant mutant M221L, pH and temperature not specified in the publication | Variovorax paradoxus | |
| 0.0196 | - |
N-acetyl-D-methionine | recombinant mutant M39L, pH and temperature not specified in the publication | Variovorax paradoxus | |
| 0.0279 | - |
N-acetyl-D-methionine | recombinant wild-type enzyme, pH and temperature not specified in the publication | Variovorax paradoxus | |
| 0.263 | - |
N-acetyl-D-methionine | recombinant mutant M56L, pH and temperature not specified in the publication | Variovorax paradoxus | |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Zn2+ | required, enzyme-bound | Variovorax paradoxus | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| N-acetyl-D-methionine + H2O | Variovorax paradoxus | - |
acetate + D-methionine | - |
? | |
| N-acetyl-D-methionine + H2O | Variovorax paradoxus Iso1 | - |
acetate + D-methionine | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Variovorax paradoxus | Q6EMR8 | - |
- |
| Variovorax paradoxus Iso1 | Q6EMR8 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant wild-type and mutant enzymes from Escherichia coli strain JM109 by ammonium sulfate fractionation, anion exchange chromatography, hydrophobic interaction chromatography, and ultrafiltration | Variovorax paradoxus |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 51.27 | - |
purified recombinant mutant M56L, substrate N-acetyl-D-methionine, pH and temperature not specified in the publication | Variovorax paradoxus |
| 52.83 | - |
purified recombinant mutant M254L, substrate N-acetyl-D-methionine, pH and temperature not specified in the publication | Variovorax paradoxus |
| 54.42 | - |
purified recombinant mutant M352L, substrate N-acetyl-D-methionine, pH and temperature not specified in the publication | Variovorax paradoxus |
| 71.06 | - |
purified recombinant mutant M39L, substrate N-acetyl-D-methionine, pH and temperature not specified in the publication | Variovorax paradoxus |
| 76.58 | - |
purified recombinant wild-type enzyme, substrate N-acetyl-D-methionine, pH and temperature not specified in the publication | Variovorax paradoxus |
| 85.78 | - |
purified recombinant mutant M221L, substrate N-acetyl-D-methionine, pH and temperature not specified in the publication | Variovorax paradoxus |
Storage Stability
| Storage Stability | Organism |
|---|---|
| the recombinant enzyme is unstable during protein purification and storage at 4°C probably due to methionine oxidation | Variovorax paradoxus |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| N-acetyl-D-methionine + H2O | - |
Variovorax paradoxus | acetate + D-methionine | - |
? | |
| N-acetyl-D-methionine + H2O | - |
Variovorax paradoxus Iso1 | acetate + D-methionine | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| N-Acyl-D-amino acid amidohydrolase | - |
Variovorax paradoxus |
| N-D-AAase | - |
Variovorax paradoxus |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 25.2 | - |
N-acetyl-D-methionine | recombinant mutant M56L, pH and temperature not specified in the publication | Variovorax paradoxus | |
| 34.56 | - |
N-acetyl-D-methionine | recombinant mutant M352L, pH and temperature not specified in the publication | Variovorax paradoxus | |
| 54.06 | - |
N-acetyl-D-methionine | recombinant mutant M254L, pH and temperature not specified in the publication | Variovorax paradoxus | |
| 57.53 | - |
N-acetyl-D-methionine | recombinant wild-type enzyme, pH and temperature not specified in the publication | Variovorax paradoxus | |
| 61.31 | - |
N-acetyl-D-methionine | recombinant mutant M39L, pH and temperature not specified in the publication | Variovorax paradoxus | |
| 76.08 | - |
N-acetyl-D-methionine | recombinant mutant M221L, pH and temperature not specified in the publication | Variovorax paradoxus | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| malfunction | replacement of methionine M221 with leucine in the enzyme successfully enhances the oxidative resistance, half-life, and enzyme activity | Variovorax paradoxus |
| additional information | structure comparison of wild-type and mutant enzyme with the enzyme from Alcaligenes faecalis DA1, homology modeling, overview | Variovorax paradoxus |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 2.06 | - |
N-acetyl-D-methionine | recombinant wild-type enzyme, pH and temperature not specified in the publication | Variovorax paradoxus | |
| 2.41 | - |
N-acetyl-D-methionine | recombinant mutant M352L, pH and temperature not specified in the publication | Variovorax paradoxus | |
| 3.12 | - |
N-acetyl-D-methionine | recombinant mutant M39L, pH and temperature not specified in the publication | Variovorax paradoxus | |
| 3.94 | - |
N-acetyl-D-methionine | recombinant mutant M254L, pH and temperature not specified in the publication | Variovorax paradoxus | |
| 4.9 | - |
N-acetyl-D-methionine | recombinant mutant M221L, pH and temperature not specified in the publication | Variovorax paradoxus | |
| 9.58 | - |
N-acetyl-D-methionine | recombinant mutant M56L, pH and temperature not specified in the publication | Variovorax paradoxus | |
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