Cloned (Comment) | Organism |
---|---|
C-terminally His6-tagged T-protein wild-type and mutant enzymes expression in Escherichia coli strain BL21(DE3)pLysS | Escherichia coli |
Crystallization (Comment) | Organism |
---|---|
aminomethyltransferase/T-protein in complex with dihydrolipoate-bearing H-protein and 5-methyltetrahydrofolate, a complex mimicking the ternary complex in the reverse reaction, purified recombinant wild-type and mutant T-proteins, hanging drop vapor diffusion method, mixing of 22-25 mg/ml proteins, T-protein and H protein, with an equal volume of reservoir solution containing 0.095 M 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid, pH 7.5, 0.19 M calcium chloride, 26.6% v/v PEG 400, and 5% v/v glycerol, at 15°C, 4-5 days to 2 weeks, soaking of the native crystals in mother liquid containing 1 mM (6S)-5-methyltetrahydrofolate at 15°C for 4 h, X-ray diffraction structure determination and analysis at 2.0 A resolution, molecular replacement | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
D96N | site-directed mutagenesis, both the glycine cleavage and synthesis activities are reduced to 34% compared to the wild-type enzyme | Escherichia coli |
D96N/Y188F | site-directed mutagenesis, the mutations abolish both the glycine cleavage and synthesis activities | Escherichia coli |
D97N | site-directed mutagenesis, the mutation abolishes both the glycine cleavage and synthesis activities | Escherichia coli |
D97N/Y188F | site-directed mutagenesis, the mutations abolish both the glycine cleavage and synthesis activities | Escherichia coli |
N113A | site-directed mutagenesis, the mutation abolishes both the glycine cleavage and synthesis activities | Escherichia coli |
N113A/R223A | site-directed mutagenesis, the mutations abolish both the glycine cleavage and synthesis activities | Escherichia coli |
N113D | site-directed mutagenesis, the mutation abolishes both the glycine cleavage and synthesis activities | Escherichia coli |
R223A | site-directed mutagenesis, the mutation abolishes both the glycine cleavage and synthesis activities | Escherichia coli |
R223K | site-directed mutagenesis, the mutation abolishes both the glycine cleavage and synthesis activities | Escherichia coli |
Y188F | site-directed mutagenesis, both the glycine cleavage and synthesis activities are reduced to 83% compared to the wild-type enzyme | Escherichia coli |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
57000 | - |
T-protein in complex with H-protein, gel filtration | Escherichia coli |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
[protein]-S8-aminomethyldihydrolipoyllysine + tetrahydrofolate | Escherichia coli | - |
[protein]-dihydrolipoyllysine + 5,10-methylenetetrahydrofolate + NH3 | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | - |
- |
- |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
[protein]-S8-aminomethyldihydrolipoyllysine + tetrahydrofolate = [protein]-dihydrolipoyllysine + 5,10-methylenetetrahydrofolate + NH3 | T-protein recognition of lipoyl protein substrate and reaction mechanism, overview. The reversible transfer of the methylene group between the lipoate and tetrahydrofolate proceeds through the electron relay-assisted iminium intermediate formation | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
[protein]-S8-aminomethyldihydrolipoyllysine + tetrahydrofolate | - |
Escherichia coli | [protein]-dihydrolipoyllysine + 5,10-methylenetetrahydrofolate + NH3 | - |
r |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Escherichia coli |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7 | - |
assay at | Escherichia coli |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
pyridoxal 5'-phosphate | - |
Escherichia coli |
General Information | Comment | Organism |
---|---|---|
metabolism | aminomethyltransferase is a component of the T-protein, which is part of a multienzyme system composed of four proteins termed P-, H-, T-, and L-protein. T-protein/aminomethyltransferase degrades the aminomethyl moiety to ammonia and 5,10-methylentetrahydrofolate in the presence of tetrahydrofolate, leaving dihydrolipoate-bearing H-protein | Escherichia coli |
additional information | T-protein in complex with dihydrolipoate-bearing H-protein and 5-methyltetrahydrofolate, a complex mimicking the ternary complex in the reverse reaction, shows a highly interacting intermolecular interface limited to a small area and the protein-bound dihydrolipoyllysine arm inserted into the active site cavity of the T-protein. Arg292 of the T-protein is essential for complex assembly | Escherichia coli |
physiological function | aminomethyltransferase reversibly catalyzes the degradation of the aminomethyl moiety of glycine attached to the lipoate cofactor of H-protein, resulting in the production of ammonia, 5,10-methylenetetrahydrofolate, and dihydrolipoate-bearing H-protein in the presence of tetrahydrofolate | Escherichia coli |