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Literature summary extracted from

  • Shaw, D.; Odom, J.D.; Dunlap, R.B.
    High expression and steady-state kinetic characterization of methionine site-directed mutants of Escherichia coli methionyl- and selenomethionyl-dihydrofolate reductase (1999), Biochim. Biophys. Acta, 1429, 401-410.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.5.1.3 high expression rate of wild-type and mutants from expression vector pCOCK Escherichia coli

Protein Variants

EC Number Protein Variants Comment Organism
1.5.1.3 L16M site-directed mutagenesis, same kinetic properties like the wild-type enzyme Escherichia coli
1.5.1.3 L16M/L20M site-directed mutagenesis, double mutant, elevated turnover number and specific activity Escherichia coli
1.5.1.3 L16M/L20M/L42M site-directed mutagenesis, triple mutant, elevated turnover number and specific activity Escherichia coli
1.5.1.3 L16SeM site-directed mutagenesis, with selenomethionine, same kinetic properties like the wild-type enzyme Escherichia coli
1.5.1.3 L16SeM/L20SeM site-directed mutagenesis, double mutant, with selenomethionine, elevated turnover number and specific activity Escherichia coli
1.5.1.3 L16SeM/L20SeM/L42SeM site-directed mutagenesis, triple mutant, with selenomethionine, elevated turnover number and specific activity Escherichia coli
1.5.1.3 L20M site-directed mutagenesis, elevated turnover number and specific activity Escherichia coli
1.5.1.3 L20SeM site-directed mutagenesis, with selenomethionine, elevated turnover number and specific activity Escherichia coli
1.5.1.3 L42M site-directed mutagenesis, same kinetic properties like the wild-type enzyme Escherichia coli
1.5.1.3 L42SeM site-directed mutagenesis, with selenomethionine, same kinetic properties like the wild-type enzyme Escherichia coli
1.5.1.3 L92M site-directed mutagenesis, same kinetic properties like the wild-type enzyme Escherichia coli
1.5.1.3 L92SeM site-directed mutagenesis, with selenomethionine, same kinetic properties like the wild-type enzyme Escherichia coli

General Stability

EC Number General Stability Organism
1.5.1.3 NADPH stabilizes Escherichia coli

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.5.1.3 additional information
-
 additional information selenomethionine-containing mutants Escherichia coli
1.5.1.3 0.00097
-
 7,8-dihydrofolate wild-type enzyme from plasmid Escherichia coli
1.5.1.3 0.00123
-
 7,8-dihydrofolate mutant L92M from plasmid Escherichia coli
1.5.1.3 0.00167
-
 7,8-dihydrofolate double mutant L16M/L20M from plasmid Escherichia coli
1.5.1.3 0.00171
-
 7,8-dihydrofolate mutant L42M from plasmid Escherichia coli
1.5.1.3 0.00222
-
 7,8-dihydrofolate mutant L16M from plasmid Escherichia coli
1.5.1.3 0.00228
-
 NADPH double mutant L16M/L20M from plasmid Escherichia coli
1.5.1.3 0.00232
-
 NADPH mutant L16M from plasmid Escherichia coli
1.5.1.3 0.00252
-
 NADPH wild-type enzyme from plasmid Escherichia coli
1.5.1.3 0.0027
-
 NADPH mutant L92M from plasmid Escherichia coli
1.5.1.3 0.00289
-
 7,8-dihydrofolate mutant L20M from plasmid Escherichia coli
1.5.1.3 0.003
-
 NADPH mutant L42M from plasmid Escherichia coli
1.5.1.3 0.00369
-
 7,8-dihydrofolate triple mutant L16M/L20M/L42M from plasmid Escherichia coli
1.5.1.3 0.0043
-
 NADPH mutant L20M from plasmid Escherichia coli
1.5.1.3 0.00456
-
 NADPH triple mutant L16M/L20M/L42M from plasmid Escherichia coli

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.5.1.3 18000
-
 gel filtration Escherichia coli

Organism

EC Number Organism UniProt Comment Textmining
1.5.1.3 Escherichia coli
-
 wild-type and mutants
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.5.1.3 wild-type and mutants from high expression vector pCOCK, large scale purification Escherichia coli

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.5.1.3 additional information
-
 selenomethionine-containing mutants Escherichia coli
1.5.1.3 45.9
-
 wild-type enzyme from plasmid Escherichia coli
1.5.1.3 90.2
-
 double mutant L16M/L20M from plasmid Escherichia coli
1.5.1.3 92.7
-
 mutant L20M from plasmid Escherichia coli
1.5.1.3 172
-
 triple mutant L16M/L20M/L42M from plasmid Escherichia coli

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.5.1.3 7,8-dihydrofolate + NADPH
-
 Escherichia coli 5,6,7,8-tetrahydrofolate + NADP+
-
 ?

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
1.5.1.3 37
-
 with NADPH bound all mutants retain activity longer than the wild-type enzyme, half-life of the wild-type enzyme 32 days Escherichia coli
1.5.1.3 37
-
 without NADPH, 50% loss of activity of wild-type and mutant enzymes after 60 h Escherichia coli

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.5.1.3 additional information
-
 additional information selenomethionine-containing mutants Escherichia coli
1.5.1.3 13.7
-
 7,8-dihydrofolate wild-type enzyme from plasmid Escherichia coli
1.5.1.3 24.6
-
 7,8-dihydrofolate mutant L20M from plasmid Escherichia coli
1.5.1.3 25.2
-
 7,8-dihydrofolate double mutant L16M/L20M from plasmid Escherichia coli
1.5.1.3 52.7
-
 7,8-dihydrofolate triple mutant L16M/L20M/L42M from plasmid Escherichia coli

Cofactor

EC Number Cofactor Comment Organism Structure
1.5.1.3 NADPH wild-type and mutant enzymes Escherichia coli