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Literature summary for 4.1.2.38 extracted from

  • Janzen, E.; Mueller, M.; Kolter-Jung, D.; Kneen, M.M.; McLeish, M.J.; Pohl, M.
    Characterization of benzaldehyde lyase from Pseudomonas fluorescens: A versatile enzyme for asymmetric C-C bond formation (2006), Bioorg. Chem., 34, 345-361.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli strain SG13009 Pseudomonas fluorescens

Protein Variants

Protein Variants Comment Organism
A28S mutant shows decarboxylase activity towards 2-keto acids Pseudomonas fluorescens
A480I reduced lyase activity Pseudomonas fluorescens
F484I reduced lyase activity Pseudomonas fluorescens
H286A reduced lyase activity Pseudomonas fluorescens

Inhibitors

Inhibitors Comment Organism Structure
Mg2+ inhibitory above 7.5 mM Pseudomonas fluorescens

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.045
-
Benzoin mutant enzyme A480I, in the presence of 15% (v/v) PEG-400, in 50 mM Tris at 30°C and pH 7.5 Pseudomonas fluorescens
0.047
-
Benzoin mutant enzyme A28S, in the presence of 15% (v/v) PEG-400, in 50 mM Tris at 30°C and pH 7.5 Pseudomonas fluorescens
0.048
-
Benzoin wild type enzyme, in the presence of 15% (v/v) PEG-400, in 50 mM Tris at 30°C and pH 7.5 Pseudomonas fluorescens
0.055
-
Benzoin mutant enzyme H286A, in the presence of 15% (v/v) PEG-400, in 50 mM Tris at 30°C and pH 7.5 Pseudomonas fluorescens
0.067
-
Benzoin mutant enzyme F484I, in the presence of 15% (v/v) PEG-400, in 50 mM Tris at 30°C and pH 7.5 Pseudomonas fluorescens
0.3
-
2-hydroxypropiophenone wild type enzyme, at 30°C and pH 7.5 Pseudomonas fluorescens
0.6
-
2-hydroxypropiophenone mutant enzyme A28S, at 30°C and pH 7.5 Pseudomonas fluorescens
0.9
-
2-hydroxypropiophenone mutant enzyme H286A, at 30°C and pH 7.5 Pseudomonas fluorescens
1.2
-
2-hydroxypropiophenone mutant enzyme F484I, at 30°C and pH 7.5 Pseudomonas fluorescens
5
-
2-hydroxypropiophenone mutant enzyme A480I, at 30°C and pH 7.5 Pseudomonas fluorescens
10
-
benzaldehyde in 50 mM potassium phosphate at 30°C and pH 7.5 Pseudomonas fluorescens

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ dependent, 1-5 mM Pseudomonas fluorescens

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
54000
-
4 * 54000, gel filtration Pseudomonas fluorescens
59800
-
His-tagged BAL subunit, SDS-PAGE and MALDI-TOF-MS Pseudomonas fluorescens
216000
-
gel filtration Pseudomonas fluorescens

Organic Solvent Stability

Organic Solvent Comment Organism
DMSO the addition of 20% (v/v) DMSO is useful to enhance both the solubility of aromatic substrates and products and the stability of BAL Pseudomonas fluorescens

Organism

Organism UniProt Comment Textmining
Pseudomonas fluorescens
-
strain biovar I
-

Purification (Commentary)

Purification (Comment) Organism
immobilized metal ion chromatography, after which the enzyme shows only 35-45% of its activity, however, activity is completely regained after incubation in the presence of Mg2+ and thiamine diphosphate Pseudomonas fluorescens

Storage Stability

Storage Stability Organism
30°C, in cofactor-free potassium phosphate buffer at pH 7.0, 3 h, 65% loss of activity Pseudomonas fluorescens

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-hydroxypropiophenone
-
Pseudomonas fluorescens ?
-
?
benzaldehyde + (R)-2,2-dimethyl-1,3-dioxolan-4-carbaldehyde
-
Pseudomonas fluorescens acyloin + benzoin
-
?
benzoin
-
Pseudomonas fluorescens benzaldehyde + benzaldehyde
-
r
additional information unable to cleave acetoin Pseudomonas fluorescens ?
-
?

Subunits

Subunits Comment Organism
tetramer 4 * 54000, gel filtration Pseudomonas fluorescens

Synonyms

Synonyms Comment Organism
benzaldehyde lyase the wild type enzyme does not show any decarboxylase activity towards 2-keto acids Pseudomonas fluorescens

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
35
-
maximal activity for the benzoin lyase reaction Pseudomonas fluorescens

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
25
-
a concentration of 1 mM MgSO4 and 0.01 mM thiamine diphosphate is sufficient to keep BAL stable at 25°C Pseudomonas fluorescens
37
-
reaction temperature shall not exceed 37°C, in the presence of 5 mM MgSO4 and 0.1 mM thiamine diphosphate the enzyme's activity is reduced by only 15% over 3 h at 37°C, however, a concentration of 1 mM MgSO4 and 0.01 mM thiamine diphosphate leads to more than 40% inactivation within 3 h at 37°C Pseudomonas fluorescens

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.6
-
2-hydroxypropiophenone mutant enzyme A28S, at 30°C and pH 7.5 Pseudomonas fluorescens
2.8
-
2-hydroxypropiophenone mutant enzyme H286A, at 30°C and pH 7.5 Pseudomonas fluorescens
3.4
-
2-hydroxypropiophenone wild type enzyme, at 30°C and pH 7.5 Pseudomonas fluorescens
4
-
2-hydroxypropiophenone mutant enzyme F484I, at 30°C and pH 7.5 Pseudomonas fluorescens
4.9
-
2-hydroxypropiophenone mutant enzyme A480I, at 30°C and pH 7.5 Pseudomonas fluorescens
6.6
-
Benzoin mutant enzyme A480I, in the presence of 15% (v/v) PEG-400, in 50 mM Tris at 30°C and pH 7.5 Pseudomonas fluorescens
9.5
-
Benzoin mutant enzyme A28S, in the presence of 15% (v/v) PEG-400, in 50 mM Tris at 30°C and pH 7.5 Pseudomonas fluorescens
22
-
Benzoin mutant enzyme F484I, in the presence of 15% (v/v) PEG-400, in 50 mM Tris at 30°C and pH 7.5 Pseudomonas fluorescens
56.4
-
Benzoin mutant enzyme H286A, in the presence of 15% (v/v) PEG-400, in 50 mM Tris at 30°C and pH 7.5 Pseudomonas fluorescens
66.9
-
Benzoin wild type enzyme, in the presence of 15% (v/v) PEG-400, in 50 mM Tris at 30°C and pH 7.5 Pseudomonas fluorescens

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
pH-optimum of both the lyase and the ligase reaction, in potassium phosphate, Tris, and imidazole buffers Pseudomonas fluorescens

pH Stability

pH Stability pH Stability Maximum Comment Organism
6 8 in potassium phosphate, Tris, and imidazole buffers, pH values below 6 lead to complete inactivation after about one day, while the enzyme shows some activity when kept above pH 8 Pseudomonas fluorescens

Cofactor

Cofactor Comment Organism Structure
thiamine diphosphate dependent, the optimal concentration is 0.1-5 mM, a significant loss of activity is observed with a concentration below 0.01 mM Pseudomonas fluorescens

pI Value

Organism Comment pI Value Maximum pI Value
Pseudomonas fluorescens isoelectric focusing
-
4.6