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Literature summary for 4.4.1.15 extracted from
- The interconversion of ACC deaminase and D-cysteine desulfhydrase by diected mutagenesis (2008), Planta, 229, 193-205.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| cloning into the pET30a (+) vector at the EcoRV/HindIII sites, all single and double mutants are constructed using a Phusion Site Directed Mutagenesis Kit. | Pseudomonas putida |
| Expression of all recombinant proteins and mutants is carried out in Escherichia coli BL21. Altering of only two amino acid residues within the predicted active site served to change the enzyme from D-cysteine desulfhydrase to ACC deaminase. | Solanum lycopersicum |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| E295S | By site-directed mutagenesis. The Pseudomonas putida UW4 single mutant is constructed using pET30a (+) with the full-length ACC deaminase as the template | Pseudomonas putida |
| E295S/L322T | The double mutant is constructed using the E295S mutant as the template. | Pseudomonas putida |
| S358E | By site-directed mutagenesis. The single mutants of the tomato enzyme are constructed using pET30 Xa/LIC with the full-length putative ACC deaminase as the template | Solanum lycopersicum |
| S358E/T386L | The double mutant is constructed using an additional round of mutagenesis and with the S358E single mutant as the template | Solanum lycopersicum |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| aminooxyacetic acid | 0.005-1 mM, enzymatic activity is progressively inhibited as the aminooxyacetic acid concentration is increased | Pseudomonas putida |  |
| aminooxyacetic acid | 0.005-1 mM, enzymatic activity is progressively inhibited as the aminooxyacetic acid concentration is increased | Solanum lycopersicum | |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.21 | - |
D-cysteine | pH 8.0 at 37°C | Solanum lycopersicum | |
| 0.34 | - |
D-cysteine | double mutant Pseudomonas putida E295S/L322T | Pseudomonas putida | |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 51480 | - |
recombinant protein is determined by mass spectrometry | Solanum lycopersicum |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| D-cysteine + H2O | Pseudomonas putida | - |
sulfide + NH3 + pyruvate | - |
? | |
| D-cysteine + H2O | Solanum lycopersicum | - |
sulfide + NH3 + pyruvate | - |
? | |
| D-cysteine + H2O | Pseudomonas putida UW4 | - |
sulfide + NH3 + pyruvate | - |
? | |
| additional information | Solanum lycopersicum | Site-directed mutagenesis shows that altering only two amino acid residues within the predicted active site served to change the enzyme from D-cysteine desulfhydrase to 1-aminocyclopropane-1-carboxylate deaminase. | ? | - |
? | |
| additional information | Pseudomonas putida | Site-directed mutagenesis shows that altering two amino acid residues at the same positions within the active site of tive site served to change the enzyme from D-cysteine desulfhydrase to deaminase from Pseudomonas putida UW4 the enzyme is converted into D-cysteine desulfhydrase. | ? | - |
? | |
| additional information | Pseudomonas putida UW4 | Site-directed mutagenesis shows that altering two amino acid residues at the same positions within the active site of tive site served to change the enzyme from D-cysteine desulfhydrase to deaminase from Pseudomonas putida UW4 the enzyme is converted into D-cysteine desulfhydrase. | ? | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Pseudomonas putida | - |
- |
- |
| Pseudomonas putida UW4 | - |
- |
- |
| Solanum lycopersicum | B2MWN0 | bony best variety | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| Recombinant protein expressed with an N-terminal 6xHis-tag is purified under native/non-denaturing conditions using Ni-NTA Superflow resin. | Pseudomonas putida |
| The recombinant protein expressed with an N-terminal 6xHis-tag is purified under native/non-denaturing conditions using Ni-NTA Superflow resin | Solanum lycopersicum |
Source Tissue
| Source Tissue | Comment | Organism | Textmining |
|---|---|---|---|
| fruit | collected at the breaker stage, which is defined as the stage where first spot of pink/red color appears at the blossom end | Solanum lycopersicum | - |
| leaf | collected at the mature green stage | Solanum lycopersicum | - |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| additional information | - |
activity below detection, D-cysteine desulfhydrase activity in double mutant S358E/T386L | Solanum lycopersicum |
| additional information | - |
activity below detection, D-cysteine desulfhydrase activity in mutant S358E. Serine at 358 residue is essential for D-cysteine desulfhydrase activity. | Solanum lycopersicum |
| 0.001594 | - |
D-cysteine desulfhydrase activity in WT | Pseudomonas putida |
| 0.0356 | - |
D-cysteine desulfhydrase activity in mutant E295S | Pseudomonas putida |
| 0.1475 | - |
D-cysteine desulfhydrase activity in double mutant E295S/L322T | Pseudomonas putida |
| 0.6653 | - |
D-cysteine desulfhydrase activity in mutant T386L. Changing the threonine 386 residue alone reduces the activity of the enzyme substantially of about 11.5% of the native recombinant enzyme, although it does not cause complete loss of activity. | Solanum lycopersicum |
| 5.784 | - |
D-cysteine desulfhydrase activity in wild-type | Solanum lycopersicum |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| D-cysteine + H2O | - |
Pseudomonas putida | sulfide + NH3 + pyruvate | - |
? | |
| D-cysteine + H2O | - |
Solanum lycopersicum | sulfide + NH3 + pyruvate | - |
? | |
| D-cysteine + H2O | - |
Pseudomonas putida UW4 | sulfide + NH3 + pyruvate | - |
? | |
| additional information | Site-directed mutagenesis shows that altering only two amino acid residues within the predicted active site served to change the enzyme from D-cysteine desulfhydrase to 1-aminocyclopropane-1-carboxylate deaminase. | Solanum lycopersicum | ? | - |
? | |
| additional information | Site-directed mutagenesis shows that altering two amino acid residues at the same positions within the active site of tive site served to change the enzyme from D-cysteine desulfhydrase to deaminase from Pseudomonas putida UW4 the enzyme is converted into D-cysteine desulfhydrase. | Pseudomonas putida | ? | - |
? | |
| additional information | Site-directed mutagenesis shows that altering two amino acid residues at the same positions within the active site of tive site served to change the enzyme from D-cysteine desulfhydrase to deaminase from Pseudomonas putida UW4 the enzyme is converted into D-cysteine desulfhydrase. | Pseudomonas putida UW4 | ? | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| D-cysteine desulfhydrase | - |
Pseudomonas putida |
| D-cysteine desulfhydrase | - |
Solanum lycopersicum |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 30 | - |
- |
Solanum lycopersicum |
| 37 | - |
assay at | Pseudomonas putida |
Temperature Stability [°C]
| Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 50 | - |
The enzyme is found to be very stable with almost 80% of its activity still remaining at 50°C | Solanum lycopersicum |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 654 | - |
D-cysteine | double mutant Pseudomonas putida E295S+L322T | Pseudomonas putida | |
| 13800 | - |
D-cysteine | - |
Solanum lycopersicum | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7.6 | - |
with estimated 6.7 (pKa1) and 8.9 (pKa2) | Solanum lycopersicum |
| 8 | - |
assay at | Pseudomonas putida |
Ki Value [mM]
| Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.0033 | - |
aminooxyacetic acid | value bases on non-linear regression | Solanum lycopersicum | |
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