BRENDA - Enzyme Database show

Identification and characterization of a highly thermostable crotonase from Meiothermus ruber

Reisse, S.; Garbe, D.; Brueck, T.; J. Mol. Catal. B 112, 40-44 (2015)
No PubMed abstract available

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
4.2.1.150
gene Mrub_2284, DNA and amino acid sequence determination and analysis, sequence comparisons and phylogenetic analysis, recombinant expression of C-terminally His-tagged enzyme in Escherichia coli strain Rosetta (DE3), subcloning in Escherichia coli strain XL-1 Blue
Meiothermus ruber
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
4.2.1.150
butanol
50% inhibition at 2% v/v, 80% at 8% v/v
Meiothermus ruber
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
4.2.1.150
0.03
-
crotonyl-CoA
pH 7.0, 50°C, recombinant His-tagged enzyme
Meiothermus ruber
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
4.2.1.150
140000
-
native PAGE, recombinant enzyme
Meiothermus ruber
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
4.2.1.150
crotonyl-CoA + H2O
Meiothermus ruber
-
3-hydroxybutyryl-CoA
-
-
r
4.2.1.150
crotonyl-CoA + H2O
Meiothermus ruber DSM 1279
-
3-hydroxybutyryl-CoA
-
-
r
Organic Solvent Stability
EC Number
Organic Solvent
Commentary
Organism
4.2.1.150
Butanol
inhibits the enzyme activity, 45% inhibition at 2% v/v, 80% at 8% v/v
Meiothermus ruber
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
4.2.1.150
Meiothermus ruber
D3PLE5
-
-
4.2.1.150
Meiothermus ruber DSM 1279
D3PLE5
-
-
Purification (Commentary)
EC Number
Commentary
Organism
4.2.1.150
recombinant His-tagged enzyme from Escherichia coli strain Rosetta (DE3) by nickel affinity chromatography and desalting gel filtration
Meiothermus ruber
Reaction
EC Number
Reaction
Commentary
Organism
4.2.1.150
a short-chain (3S)-3-hydroxyacyl-CoA = a short-chain trans-2-enoyl-CoA + H2O
the Mr-Crt catalytic mechanism constitutes a concerted attack by the two glutamate residues. While Glu143 of Mr-Crt protonates the substrate, Glu123 abstracts a proton from a bound water molecule. The Gly120 activates the substrate by a hydrogen bond to the oxygen of the enoyl moiety of the CoA ester. The binding pocket for the CoA moiety is formed by characteristic hydrophobic amino acids and lysine residues
Meiothermus ruber
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
4.2.1.150
crotonyl-CoA + H2O
-
748405
Meiothermus ruber
3-hydroxybutyryl-CoA
-
-
-
r
4.2.1.150
crotonyl-CoA + H2O
-
748405
Meiothermus ruber DSM 1279
3-hydroxybutyryl-CoA
-
-
-
r
Subunits
EC Number
Subunits
Commentary
Organism
4.2.1.150
homotetramer
4 * 30000, about, sequence calculation
Meiothermus ruber
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
4.2.1.150
55
-
-
Meiothermus ruber
Temperature Range [°C]
EC Number
Temperature Minimum [°C]
Temperature Maximum [°C]
Commentary
Organism
4.2.1.150
30
78
activity range, 20% of maximal activity at 30°C, 60% at 75°C, over 80% at 50-70°C, and maximal activity at 55°C
Meiothermus ruber
Temperature Stability [°C]
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
4.2.1.150
50
70
purified recombinant His-tagged enzyme, 80% activity remaining after 5 min
Meiothermus ruber
4.2.1.150
50
-
the purified recombinant His-tagged enzyme Mr-Crt displays an extended half-life of over 1 month, IT50: 743 h
Meiothermus ruber
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
4.2.1.150
14.9
-
crotonyl-CoA
pH 7.0, 50°C, recombinant His-tagged enzyme
Meiothermus ruber
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
4.2.1.150
7
-
-
Meiothermus ruber
pH Range
EC Number
pH Minimum
pH Maximum
Commentary
Organism
4.2.1.150
6.5
7.5
60% of maximal activity is observed at pH 6.5 and pH 7.5
Meiothermus ruber
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
4.2.1.150
gene Mrub_2284, DNA and amino acid sequence determination and analysis, sequence comparisons and phylogenetic analysis, recombinant expression of C-terminally His-tagged enzyme in Escherichia coli strain Rosetta (DE3), subcloning in Escherichia coli strain XL-1 Blue
Meiothermus ruber
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
4.2.1.150
butanol
50% inhibition at 2% v/v, 80% at 8% v/v
Meiothermus ruber
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
4.2.1.150
0.03
-
crotonyl-CoA
pH 7.0, 50°C, recombinant His-tagged enzyme
Meiothermus ruber
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
4.2.1.150
140000
-
native PAGE, recombinant enzyme
Meiothermus ruber
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
4.2.1.150
crotonyl-CoA + H2O
Meiothermus ruber
-
3-hydroxybutyryl-CoA
-
-
r
4.2.1.150
crotonyl-CoA + H2O
Meiothermus ruber DSM 1279
-
3-hydroxybutyryl-CoA
-
-
r
Organic Solvent Stability (protein specific)
EC Number
Organic Solvent
Commentary
Organism
4.2.1.150
Butanol
inhibits the enzyme activity, 45% inhibition at 2% v/v, 80% at 8% v/v
Meiothermus ruber
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
4.2.1.150
recombinant His-tagged enzyme from Escherichia coli strain Rosetta (DE3) by nickel affinity chromatography and desalting gel filtration
Meiothermus ruber
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
4.2.1.150
crotonyl-CoA + H2O
-
748405
Meiothermus ruber
3-hydroxybutyryl-CoA
-
-
-
r
4.2.1.150
crotonyl-CoA + H2O
-
748405
Meiothermus ruber DSM 1279
3-hydroxybutyryl-CoA
-
-
-
r
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
4.2.1.150
homotetramer
4 * 30000, about, sequence calculation
Meiothermus ruber
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
4.2.1.150
55
-
-
Meiothermus ruber
Temperature Range [°C] (protein specific)
EC Number
Temperature Minimum [°C]
Temperature Maximum [°C]
Commentary
Organism
4.2.1.150
30
78
activity range, 20% of maximal activity at 30°C, 60% at 75°C, over 80% at 50-70°C, and maximal activity at 55°C
Meiothermus ruber
Temperature Stability [°C] (protein specific)
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
4.2.1.150
50
70
purified recombinant His-tagged enzyme, 80% activity remaining after 5 min
Meiothermus ruber
4.2.1.150
50
-
the purified recombinant His-tagged enzyme Mr-Crt displays an extended half-life of over 1 month, IT50: 743 h
Meiothermus ruber
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
4.2.1.150
14.9
-
crotonyl-CoA
pH 7.0, 50°C, recombinant His-tagged enzyme
Meiothermus ruber
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
4.2.1.150
7
-
-
Meiothermus ruber
pH Range (protein specific)
EC Number
pH Minimum
pH Maximum
Commentary
Organism
4.2.1.150
6.5
7.5
60% of maximal activity is observed at pH 6.5 and pH 7.5
Meiothermus ruber
General Information
EC Number
General Information
Commentary
Organism
4.2.1.150
additional information
The Mr-Crt catalytic triad is formed by residues Gly120, Glu123 and Glu143
Meiothermus ruber
General Information (protein specific)
EC Number
General Information
Commentary
Organism
4.2.1.150
additional information
The Mr-Crt catalytic triad is formed by residues Gly120, Glu123 and Glu143
Meiothermus ruber
KCat/KM [mM/s]
EC Number
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
4.2.1.150
496.7
-
crotonyl-CoA
pH 7.0, 50°C, recombinant His-tagged enzyme
Meiothermus ruber
KCat/KM [mM/s] (protein specific)
EC Number
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
4.2.1.150
496.7
-
crotonyl-CoA
pH 7.0, 50°C, recombinant His-tagged enzyme
Meiothermus ruber