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Literature summary extracted from

  • Todorovic, B.; Glick, B.R.
    The interconversion of ACC deaminase and D-cysteine desulfhydrase by diected mutagenesis (2008), Planta, 229, 193-205.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.5.99.7 Pseudomonas putida UW4 ACC deaminase is cloned 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
4.4.1.15 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
4.4.1.15 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

EC Number Protein Variants Comment Organism
3.5.99.7 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
3.5.99.7 E295S/L322T the double mutant is constructed using the E295S mutant as the template Pseudomonas putida
4.4.1.15 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
4.4.1.15 E295S/L322T The double mutant is constructed using the E295S mutant as the template. Pseudomonas putida
4.4.1.15 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
4.4.1.15 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

EC Number Inhibitors Comment Organism Structure
3.5.99.7 aminooxyacetic acid 0.005-1 mM, enzymatic activity is progressively inhibited as the aminooxyacetic acid concentration is increased Pseudomonas putida
4.4.1.15 aminooxyacetic acid 0.005-1 mM, enzymatic activity is progressively inhibited as the aminooxyacetic acid concentration is increased Pseudomonas putida
4.4.1.15 aminooxyacetic acid 0.005-1 mM, enzymatic activity is progressively inhibited as the aminooxyacetic acid concentration is increased Solanum lycopersicum

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.5.99.7 0.34
-
D-cysteine double mutant Pseudomonas putida E295S+L322T Pseudomonas putida
4.4.1.15 0.21
-
D-cysteine pH 8.0 at 37°C Solanum lycopersicum
4.4.1.15 0.34
-
D-cysteine double mutant Pseudomonas putida E295S/L322T Pseudomonas putida

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
4.4.1.15 51480
-
recombinant protein is determined by mass spectrometry Solanum lycopersicum

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.5.99.7 1-aminocyclopropane-1-carboxylate + H2O Pseudomonas putida activity of the mutants, immediate precursor of ethylene in plants alpha-ketobutyrate + NH3
-
?
3.5.99.7 1-aminocyclopropane-1-carboxylate + H2O Pseudomonas putida UW4 activity of the mutants, immediate precursor of ethylene in plants alpha-ketobutyrate + NH3
-
?
3.5.99.7 D-cysteine + H2O Pseudomonas putida
-
sulfide + NH3 + pyruvate
-
?
3.5.99.7 D-cysteine + H2O Pseudomonas putida UW4
-
sulfide + NH3 + pyruvate
-
?
3.5.99.7 additional information Pseudomonas putida Site-directed mutagenesis shows that altering two amino acid residues at the same positions within the predicted active site serves to change the enzyme from D-cysteine desulfhydrase to deaminase from Pseudomonas putida UW4 the enzyme is converted into D-cysteine desulfhydrase. ?
-
?
3.5.99.7 additional information Pseudomonas putida UW4 Site-directed mutagenesis shows that altering two amino acid residues at the same positions within the predicted active site serves to change the enzyme from D-cysteine desulfhydrase to deaminase from Pseudomonas putida UW4 the enzyme is converted into D-cysteine desulfhydrase. ?
-
?
4.4.1.15 D-cysteine + H2O Pseudomonas putida
-
sulfide + NH3 + pyruvate
-
?
4.4.1.15 D-cysteine + H2O Solanum lycopersicum
-
sulfide + NH3 + pyruvate
-
?
4.4.1.15 D-cysteine + H2O Pseudomonas putida UW4
-
sulfide + NH3 + pyruvate
-
?
4.4.1.15 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. ?
-
?
4.4.1.15 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. ?
-
?
4.4.1.15 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

EC Number Organism UniProt Comment Textmining
3.5.99.7 Pseudomonas putida
-
-
-
4.4.1.15 Pseudomonas putida
-
-
-
4.4.1.15 Pseudomonas putida UW4
-
-
-
4.4.1.15 Solanum lycopersicum B2MWN0 bony best variety
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.5.99.7 Recombinant protein expressed with an N-terminal 6xHis-tag is purified under native/non-denaturing conditions using Ni-NTA Superflow resin. Pseudomonas putida
4.4.1.15 Recombinant protein expressed with an N-terminal 6xHis-tag is purified under native/non-denaturing conditions using Ni-NTA Superflow resin. Pseudomonas putida
4.4.1.15 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

EC Number Source Tissue Comment Organism Textmining
3.5.99.7 cell culture strain UW4 Pseudomonas putida
-
4.4.1.15 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
-
4.4.1.15 leaf collected at the mature green stage Solanum lycopersicum
-

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
3.5.99.7 additional information
-
activity below detection, ACC deaminase activity in double mutant E295S/L322T Pseudomonas putida
3.5.99.7 0.001594
-
D-cysteine desulfhydrase activity in wild-type enzyme Pseudomonas putida
3.5.99.7 0.00819
-
ACC deaminase activity in mutant E295S Pseudomonas putida
3.5.99.7 0.0356
-
D-cysteine desulfhydrase activity in mutant E295S Pseudomonas putida
3.5.99.7 0.1475
-
D-cysteine desulfhydrase activity in double mutant E295S+L322T Pseudomonas putida
3.5.99.7 2.812
-
ACC deaminase activity in wild-type enzyme Pseudomonas putida
4.4.1.15 additional information
-
activity below detection, D-cysteine desulfhydrase activity in double mutant S358E/T386L Solanum lycopersicum
4.4.1.15 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
4.4.1.15 0.001594
-
D-cysteine desulfhydrase activity in WT Pseudomonas putida
4.4.1.15 0.0356
-
D-cysteine desulfhydrase activity in mutant E295S Pseudomonas putida
4.4.1.15 0.1475
-
D-cysteine desulfhydrase activity in double mutant E295S/L322T Pseudomonas putida
4.4.1.15 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
4.4.1.15 5.784
-
D-cysteine desulfhydrase activity in wild-type Solanum lycopersicum

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.5.99.7 1-aminocyclopropane-1-carboxylate + H2O activity of the mutants, immediate precursor of ethylene in plants Pseudomonas putida alpha-ketobutyrate + NH3
-
?
3.5.99.7 1-aminocyclopropane-1-carboxylate + H2O activity of the mutants, immediate precursor of ethylene in plants Pseudomonas putida UW4 alpha-ketobutyrate + NH3
-
?
3.5.99.7 D-cysteine + H2O
-
Pseudomonas putida sulfide + NH3 + pyruvate
-
?
3.5.99.7 D-cysteine + H2O
-
Pseudomonas putida UW4 sulfide + NH3 + pyruvate
-
?
3.5.99.7 additional information Site-directed mutagenesis shows that altering two amino acid residues at the same positions within the predicted active site serves to change the enzyme from D-cysteine desulfhydrase to deaminase from Pseudomonas putida UW4 the enzyme is converted into D-cysteine desulfhydrase. Pseudomonas putida ?
-
?
3.5.99.7 additional information Site-directed mutagenesis shows that altering two amino acid residues at the same positions within the predicted active site serves 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 ?
-
?
4.4.1.15 D-cysteine + H2O
-
Pseudomonas putida sulfide + NH3 + pyruvate
-
?
4.4.1.15 D-cysteine + H2O
-
Solanum lycopersicum sulfide + NH3 + pyruvate
-
?
4.4.1.15 D-cysteine + H2O
-
Pseudomonas putida UW4 sulfide + NH3 + pyruvate
-
?
4.4.1.15 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 ?
-
?
4.4.1.15 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 ?
-
?
4.4.1.15 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

EC Number Synonyms Comment Organism
3.5.99.7 ACC deaminase
-
Pseudomonas putida
4.4.1.15 D-cysteine desulfhydrase
-
Pseudomonas putida
4.4.1.15 D-cysteine desulfhydrase
-
Solanum lycopersicum

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.5.99.7 37
-
assay at Pseudomonas putida
4.4.1.15 30
-
-
Solanum lycopersicum
4.4.1.15 37
-
assay at Pseudomonas putida

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
4.4.1.15 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]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.5.99.7 654
-
D-cysteine double mutant Pseudomonas putida E295S+L322T Pseudomonas putida
4.4.1.15 654
-
D-cysteine double mutant Pseudomonas putida E295S+L322T Pseudomonas putida
4.4.1.15 13800
-
D-cysteine
-
Solanum lycopersicum

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.5.99.7 8
-
assay at Pseudomonas putida
4.4.1.15 7.6
-
with estimated 6.7 (pKa1) and 8.9 (pKa2) Solanum lycopersicum
4.4.1.15 8
-
assay at Pseudomonas putida

Cofactor

EC Number Cofactor Comment Organism Structure
3.5.99.7 pyridoxal 5'-phosphate ACC deaminase binds one pyridoxal phosphate (PLP) molecule at each subunit via a conserved lysine residue. Pseudomonas putida

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
4.4.1.15 0.0033
-
aminooxyacetic acid value bases on non-linear regression Solanum lycopersicum