Literature summary for 2.1.2.10 extracted from

Okamura-Ikeda, K.; Hosaka, H.; Maita, N.; Fujiwara, K.; Yoshizawa, A.C.; Nakagawa, A.; Taniguchi, H.
Crystal structure of aminomethyltransferase in complex with dihydrolipoyl-H-protein of the glycine cleavage system: implications for recognition of lipoyl protein substrate, disease-related mutations, and reaction mechanism (2010), J. Biol. Chem., 285, 18684-18692.

Cloned(Commentary)

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 (Commentary)

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

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

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 [Da]	Molecular Weight Maximum [Da]	Comment	Organism
57000	-	T-protein in complex with H-protein, gel filtration	Escherichia coli

Natural Substrates/ Products (Substrates)

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

Organism	UniProt	Comment	Textmining
Escherichia coli	-	-	-

Reaction

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	a

Substrates and Products (Substrate)

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 [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Escherichia coli

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7	-	assay at	Escherichia coli

Cofactor

Cofactor	Comment	Organism	Structure
pyridoxal 5'-phosphate	-	Escherichia coli

General Information

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