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Literature summary for 1.5.1.3 extracted from

  • Lim, S.I.; Mizuta, Y.; Takasu, A.; Kim, Y.H.; Kwon, I.
    Site-specific bioconjugation of a murine dihydrofolate reductase enzyme by copper(I)-catalyzed azide-alkyne cycloaddition with retained activity (2014), PLoS ONE, 9, e98403 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of His-tagged wild-type mDHFR and modified mDHFR-43pEthF enzymes in Escherichia coli strain XL1-Blue Mus musculus

Protein Variants

Protein Variants Comment Organism
additional information incorporation of an alkyne-bearing non-natural amino acid, p-ethynylphenylalanine, into murine dihydrofolate reductase (mDHFR) at Val43, in high cell density cultivation of Escherichia coli, and performance of Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) conjugation with fluorescent azide dyes to evaluate enzyme compatibility with various CuAAC conditions comprising combination of commercially available Cu(I)-chelating ligands and reductants. The condensed culture improves the protein yield 19fold based on the same amount of non-natural amino acid. Enzyme incubation under optimized reaction condition does not lead to any activity loss but allows a fast and high-yield bioconjugation. Using the established conditions, a biotin-azide spacer is efficiently conjugated to enzyme mDHFR with retained activity leading to the site-specific immobilization of the biotin-conjugated mDHFR on a streptavidin-coated plate. Method evaluation, overview Mus musculus

Inhibitors

Inhibitors Comment Organism Structure
DMSO irreversibly impairs the enzymatic activity at 30% v/v Mus musculus

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
7,8-dihydrofolate + NADPH + H+ Mus musculus
-
5,6,7,8-tetrahydrofolate + NADP+
-
r

Organic Solvent Stability

Organic Solvent Comment Organism
DMSO irreversibly impairs the enzymatic activity at 30% v/v Mus musculus

Organism

Organism UniProt Comment Textmining
Mus musculus P00375
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type mDHFR and modified mDHFR-43pEthF enzymes from Escherichia coli strain XL1-Blue by nickel affinity chromatography Mus musculus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
7,8-dihydrofolate + NADPH + H+
-
Mus musculus 5,6,7,8-tetrahydrofolate + NADP+
-
r

Synonyms

Synonyms Comment Organism
dihydrofolate reductase
-
Mus musculus
mDHFR
-
Mus musculus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Mus musculus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5 8 assay at Mus musculus

Cofactor

Cofactor Comment Organism Structure
NADP+
-
Mus musculus
NADPH
-
Mus musculus