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Literature summary for 3.5.99.7 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
Search for more literature for 3.5.99.7

Cloned(Commentary)

Cloned (Comment) Organism
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

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

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

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.34
-
 D-cysteine double mutant Pseudomonas putida E295S+L322T Pseudomonas putida

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1-aminocyclopropane-1-carboxylate + H2O Pseudomonas putida activity of the mutants, immediate precursor of ethylene in plants alpha-ketobutyrate + NH3
-
 ?
1-aminocyclopropane-1-carboxylate + H2O Pseudomonas putida UW4 activity of the mutants, immediate precursor of ethylene in plants alpha-ketobutyrate + NH3
-
 ?
D-cysteine + H2O Pseudomonas putida
-
 sulfide + NH3 + pyruvate
-
 ?
D-cysteine + H2O Pseudomonas putida UW4
-
 sulfide + NH3 + pyruvate
-
 ?
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. ?
-
 ?
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. ?
-
 ?

Organism

Organism UniProt Comment Textmining
Pseudomonas putida
-
-
-

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

Source Tissue

Source Tissue Comment Organism Textmining
cell culture strain UW4 Pseudomonas putida
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
 activity below detection, ACC deaminase activity in double mutant E295S/L322T Pseudomonas putida
0.001594
-
 D-cysteine desulfhydrase activity in wild-type enzyme Pseudomonas putida
0.00819
-
 ACC deaminase activity in mutant E295S 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
2.812
-
 ACC deaminase activity in wild-type enzyme Pseudomonas putida

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1-aminocyclopropane-1-carboxylate + H2O activity of the mutants, immediate precursor of ethylene in plants Pseudomonas putida alpha-ketobutyrate + NH3
-
 ?
1-aminocyclopropane-1-carboxylate + H2O activity of the mutants, immediate precursor of ethylene in plants Pseudomonas putida UW4 alpha-ketobutyrate + NH3
-
 ?
D-cysteine + H2O
-
 Pseudomonas putida sulfide + NH3 + pyruvate
-
 ?
D-cysteine + H2O
-
 Pseudomonas putida UW4 sulfide + NH3 + pyruvate
-
 ?
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 ?
-
 ?
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 ?
-
 ?

Synonyms

Synonyms Comment Organism
ACC deaminase
-
 Pseudomonas putida

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Pseudomonas putida

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

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
 assay at Pseudomonas putida

Cofactor

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