Literature summary for 6.3.3.2 extracted from

Chen, S.; Yakunin, A.F.; Proudfoot, M.; Kim, R.; Kim, S.H.
Structural and functional characterization of a 5,10-methenyltetrahydrofolate synthetase from Mycoplasma pneumoniae (GI: 13508087) (2005), Proteins, 61, 433-443.

Cloned(Commentary)

Cloned (Comment)	Organism
overexpression of His-tagged enzyme in Escherichia coli strain BL21(DE3)	Mycoplasma pneumoniae

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant enzyme complexed with ADP, phosphate, and 5-formyltetrahydrofolate, hanging drop vapour diffusion method, 20°C, 30 mg/ml protein with 20% PEG 4000, 5 mM ATP, and 10 mM 5-formylTHF, flash freezing of all crystals in 20% PEG 4000 and 30% ethylene glycol, X-ray diffraction structure determination and analysis at 2.5 A resolution, crystallization of the enzyme with ATP analogues ATP-gamma-S, AMP-PNP, or AMP-PCP is not successful	Mycoplasma pneumoniae

Crystallization (Comment)

Organism

purified recombinant enzyme complexed with ADP, phosphate, and 5-formyltetrahydrofolate, hanging drop vapour diffusion method, 20°C, 30 mg/ml protein with 20% PEG 4000, 5 mM ATP, and 10 mM 5-formylTHF, flash freezing of all crystals in 20% PEG 4000 and 30% ethylene glycol, X-ray diffraction structure determination and analysis at 2.5 A resolution, crystallization of the enzyme with ATP analogues ATP-gamma-S, AMP-PNP, or AMP-PCP is not successful

Mycoplasma pneumoniae

Inhibitors

Inhibitors	Comment	Organism	Structure
phosphate	product inhibition	Mycoplasma pneumoniae

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	steady-state kinetics, recombinant enzyme	Mycoplasma pneumoniae
0.165	-	5-formyltetrahydrofolate	pH 6.0, 37°C, recombinant enzyme	Mycoplasma pneumoniae
0.166	-	ATP	pH 6.0, 37°C, recombinant enzyme	Mycoplasma pneumoniae

Metals/Ions

Metals/Ions	Comment	Organism
Ca2+	activates, can substitute for Mg2+	Mycoplasma pneumoniae
Co2+	activates, can substitute for Mg2+	Mycoplasma pneumoniae
Cu2+	activates, can substitute for Mg2+	Mycoplasma pneumoniae
Fe2+	activates, can substitute for Mg2+	Mycoplasma pneumoniae
Mg2+	dependent on, most effective divalent cation, required for MgATP2- complex formation, binding structure	Mycoplasma pneumoniae
Mn2+	activates, can substitute for Mg2+	Mycoplasma pneumoniae
additional information	the enzyme is dependent on divalent cation, Mg2+ is preferred	Mycoplasma pneumoniae
Zn2+	activates, can substitute for Mg2+	Mycoplasma pneumoniae

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
22000	-	monomeric recombinant enzyme, gel filtration	Mycoplasma pneumoniae
44000	-	dimeric recombinant enzyme, gel filtration	Mycoplasma pneumoniae

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ATP + 5-formyltetrahydrofolate	Mycoplasma pneumoniae	-	ADP + 5,10-methenyltetrahydrofolate + phosphate	-	?

Organism

Organism	UniProt	Comment	Textmining
Mycoplasma pneumoniae	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by metal affinity chromatography to over 95% homogeneity	Mycoplasma pneumoniae

Reaction

Reaction	Comment	Organism	Reaction ID
ATP + 5-formyltetrahydrofolate = ADP + phosphate + 5,10-methenyltetrahydrofolate	active site and substrate binding site structure, binding and catalytic mechanism	Mycoplasma pneumoniae	a

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
2.8	-	purified recombinant enzyme	Mycoplasma pneumoniae

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
ATP + 5-formyltetrahydrofolate	ATP in form of MgATP2-	Mycoplasma pneumoniae	ADP + phosphate + 5,10-methenyltetrahydrofolate	-	?
ATP + 5-formyltetrahydrofolate	-	Mycoplasma pneumoniae	ADP + 5,10-methenyltetrahydrofolate + phosphate	-	?

Subunits

Subunits	Comment	Organism
dimer	2 * 22000, recombinant enzyme, SDS-PAGE, the recombinant enyme exists as a mixture of monomers and dimers	Mycoplasma pneumoniae
monomer	1 * 22000, recombinant enzyme, SDS-PAGE, the recombinant enyme exists as a mixture of monomers and dimers	Mycoplasma pneumoniae

Synonyms

Synonyms	Comment	Organism
5,10-Methenyltetrahydrofolate synthetase	-	Mycoplasma pneumoniae
Methenyltetrahydrofolate synthetase	-	Mycoplasma pneumoniae
More	the enzyme belongs to the cycloligase protein family	Mycoplasma pneumoniae
MTHFS	-	Mycoplasma pneumoniae

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Mycoplasma pneumoniae

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
0.94	-	5-formyltetrahydrofolate	pH 6.0, 37°C, recombinant enzyme	Mycoplasma pneumoniae
0.99	-	ATP	pH 6.0, 37°C, recombinant enzyme	Mycoplasma pneumoniae

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
6	-	assay at	Mycoplasma pneumoniae

Cofactor

Cofactor	Comment	Organism	Structure
ATP	absolutely required, as MgATP2-, binding structure	Mycoplasma pneumoniae
additional information	no activity with nucleoside 5'-mono- or 5'-diphosphates	Mycoplasma pneumoniae

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism	Structure
2.12	-	phosphate	pH 6.0, 37°C, recombinant enzyme	Mycoplasma pneumoniae