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Literature summary for 1.20.1.1 extracted from

  • Woodyer, R.; van der Donk, W.A.; Zhao, H.
    Relaxing the nicotinamide cofactor specificity of phosphite dehydrogenase by rational design (2003), Biochemistry, 42, 11604-11614.
    View publication on PubMed

Application

Application Comment Organism
synthesis production of deuterium- or tritium-labeled substances, mutant enzymes could be applied as NADPH regeneration systems Pseudomonas stutzeri

Cloned(Commentary)

Cloned (Comment) Organism
wild type and mutant proteins expressed in Escherichia coli BL21(DE3) as His-tag fusion protein Pseudomonas stutzeri

Protein Variants

Protein Variants Comment Organism
A176R strongly decreased Km for NADP+ Pseudomonas stutzeri
E175A strongly decreased Km for NADP+ Pseudomonas stutzeri
E175A/A176R double mutant, 3.6-fold higher efficiency with NAD+, 1000-fold higher efficiency with NADP+, 3-fold favor for NADP+ over NAD+ as cofactor Pseudomonas stutzeri

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.004
-
NADP+ 25°C, pH 7.25, E175A/A176R mutant enzyme Pseudomonas stutzeri
0.016
-
NAD+ 25°C, pH 7.25, E175A mutant enzyme Pseudomonas stutzeri
0.02
-
NAD+ 25°C, pH 7.25, E175A/A176R mutant enzyme Pseudomonas stutzeri
0.053
-
NAD+ 25°C, pH 7.25, wild type enzyme Pseudomonas stutzeri
0.06
-
NAD+ 25°C, pH 7.25, A176R mutant enzyme Pseudomonas stutzeri
0.077
-
NADP+ 25°C, pH 7.25, A176R mutant enzyme Pseudomonas stutzeri
0.144
-
NADP+ 25°C, pH 7.25, E175A mutant enzyme Pseudomonas stutzeri
2.51
-
NADP+ 25°C, pH 7.25, wild type enzyme Pseudomonas stutzeri

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
38500
-
x * 38500, SDS-PAGE, His-tag fusion protein Pseudomonas stutzeri

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
phosphonate + H2O + NAD+ Pseudomonas stutzeri NADH regeneration system phosphate + NADH
-
ir

Organism

Organism UniProt Comment Textmining
Pseudomonas stutzeri
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant enzymes using His-tag Pseudomonas stutzeri

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
phosphonate + H2O + NAD+
-
Pseudomonas stutzeri phosphate + NADH
-
ir
phosphonate + H2O + NAD+ NADH regeneration system Pseudomonas stutzeri phosphate + NADH
-
ir
phosphonate + H2O + NADP+ 100-fold lower activity than with NAD+ Pseudomonas stutzeri phosphate + NADPH
-
ir

Subunits

Subunits Comment Organism
? x * 38500, SDS-PAGE, His-tag fusion protein Pseudomonas stutzeri

Synonyms

Synonyms Comment Organism
PTDH
-
Pseudomonas stutzeri

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
40.5
-
rapid inactivation, half life less than 10 min, 1 mM NAD+ partially protects against thermal inactivation, E175A/A176R double mutant is readily protected against inactivation by addition of 1 mM NADP+ for at least 15 min Pseudomonas stutzeri

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.41
-
NADP+ 25°C, pH 7.25, wild type enzyme Pseudomonas stutzeri
1.9
-
NADP+ 25°C, pH 7.25, E175A/A176R mutant enzyme Pseudomonas stutzeri
2.18
-
NADP+ 25°C, pH 7.25, A176R mutant enzyme Pseudomonas stutzeri
2.18
-
NADP+ 25°C, pH 7.25, E175A mutant enzyme Pseudomonas stutzeri
2.93
-
NAD+ 25°C, pH 7.25, wild type enzyme Pseudomonas stutzeri
3.5
-
NAD+ 25°C, pH 7.25, E175A mutant enzyme Pseudomonas stutzeri
3.94
-
NAD+ 25°C, pH 7.25, E175A/A176R mutant enzyme Pseudomonas stutzeri
4.28
-
NAD+ 25°C, pH 7.25, A176R mutant enzyme Pseudomonas stutzeri

Cofactor

Cofactor Comment Organism Structure
NAD+
-
Pseudomonas stutzeri
NADP+ 100-fold lower activity than with NAD+ Pseudomonas stutzeri

pI Value

Organism Comment pI Value Maximum pI Value
Pseudomonas stutzeri isoelectric focusing, wild type enzyme
-
5.8
Pseudomonas stutzeri isoelectric focusing, A176R mutant enzyme
-
6.2
Pseudomonas stutzeri isoelectric focusing, E175A mutant enzyme
-
6.2
Pseudomonas stutzeri isoelectric focusing, E175A/A176R mutant enzyme
-
6.6