Purification and properties of 5,10-methenyltetrahydrofolate synthetase from Lactobacillus casei.

Grimshaw, Charles E.; Henderson, George; Soppe, G G; Hansen, Gunda; Mathur, Eric J.; Huennekens, F.M.

doi:10.1016/s0021-9258(17)43206-6

Cited by 35 publications

(2 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This conclusion was made because alterations in the rates of decrease in A 356 produced by ascorbate, DTE, DFX, and EDTA (Figures 1-4), the recovery of nearly quantitative amounts of 5,10-CHdH 4 F after acidification only in the reactions containing ascorbate, DTE, and DFX (Figures 1-3), the presence of isosbestic points for the formation of 5-HCO-H 4 F from 5,10-CHdH 4 F only in the DTE and DFX containing reactions (Figures 5 and 6), and the chromatographic separation of 5-HCO-H 4 F and 5,10-CHdH 4 F (Figure 8). The isosbestic points for the enzyme-catalyzed conversion of 5-HCO-H 4 F to 5,10-CHdH 4 F were reported to be 258 and 318 nm (pH 6.0), which are similar to the findings reported here (15). Beavon and Blair (14) reported that 5-HCO-H 4 F is dehydrated only to 5,10-CHdH 4 F at pH 2-3.…”

Section: Discussionsupporting

confidence: 85%

Hydrolysis of 5,10-Methenyltetrahydrofolate to 5-Formyltetrahydrofolate at pH 2.5 to 4.5

Baggott

2000

Biochemistry

View full text Add to dashboard Cite

At pH 4.0 to 4.5, 5,10-methenyltetrahydrofolate is hydrolyzed to only 5-formyltetrahydrofolate if reducing agents are present or iron-redox cycling is suppressed. At pH 4.0, the equilibrium position for this hydrolysis is approximately equal concentrations of both folates. If no reducing agents are used or iron-redox cycling is promoted, considerable amounts of 10-formyldihydrofolate are also formed. It is likely that 10-formyldihydrofolate has been misidentified as 5,10-hydroxymethylenetetrahydrofolate, which was reported to accumulate during the hydrolysis of 5, 10-methenyltetrahydrofolate to 5-formyltetrahydrofolate [Stover, P. and Schirch, V. (1992) Biochemistry 31, 2148-2155 and 2155-2164; (1990) J. Biol. Chem. 265, 14227-14233]. Since 5, 10-hydroxymethylenetetrahydrofolate is reported to be the viable in vivo substrate for serine hydroxymethyltransferase-catalyzed formation of 5-formyltetrahydrofolate, and 5, 10-hydroxymethylenetetrahydrofolate probably does not accumulate, the above folate metabolism is now doubtful. It is hypothesized that mildly acidic subcellular organelles provide an environment for the hydrolysis of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate in vivo, and there is no requirement for enzyme catalysis. Finally, 10-formyltetrahydrofolate is susceptible to iron-catalyzed oxidation to 10-formyldihydrofolate at pH 4 to 4.5.

show abstract

Section: Discussionsupporting

confidence: 85%

Hydrolysis of 5,10-Methenyltetrahydrofolate to 5-Formyltetrahydrofolate at pH 2.5 to 4.5

Baggott

2000

Biochemistry

View full text Add to dashboard Cite

show abstract

“…[3,4-3 H]Glutamate (41 Ci/mmol from Moravek) was used to make the tritium-labeled polyglutamate derivatives of 5,8dideazafolate. The 5,8-dideazafolate affinity column was prepared by the method of Grimshaw et al (1984).…”

Section: Methodsmentioning

confidence: 99%

Properties of Tetrahydropteroylpentaglutamate Bound to 10-Formyltetrahydrofolate Dehydrogenase

et al. 1996

View full text Add to dashboard Cite

A new rapid procedure for purifying 10-formyltetrahydrofolate dehydrogenase results in 90 mg of pure enzyme from two rabbit livers. This abundant liver enzyme is known to bind its product tetrahydropteroylpentaglutamate (H4PteGlu5) so tightly that it does not dissociate during size exclusion chromatography. 10-Formyltetrahydrofolate dehydrogenase is also known to exhibit strong product inhibition by H4PteGlu5. There is a several-fold excess of 10-formyltetrahydrofolate dehydrogenase subunits in liver relative to the concentration of H4PteGlun, suggesting that in vivo this enzyme may bind significant amounts of this coenzyme in a nearly irreversible enzyme. H4PteGlu5 complex. How this tightly bound H4PteGlun is transferred to the other two enzymes in the cytosol, serine hydroxymethyltransferase and C1-tetrahydrofolate synthase, which use H4PteGlu5 as a substrate, is the subject of this investigation. Analysis of the product inhibition curve for 10-formyltetrahydrofolate dehydrogenase shows that H4-PteGlu5 has a dissociation constant near 15 nM which is 60-fold lower than the Ks for 10-formyl-H4PteGlu5. Fluorescence titration studies also yield a Kd of about 20 nM for H4PteGlu5. Coupling the 10-formyltetrahydrofolate dehydrogenase reaction to an excess of either serine hydroxymethyltransferase or C1-tetrahydrofolate synthase not only abolishes product inhibition but also increases the initial rate of its activity by about 2-fold. Passage of a reaction mixture of 10-formyltetrahydrofolate dehydrogenase down a size exclusion column results in enzyme with 1 equiv of H4PteGlu5 bound per subunit. However, addition of either serine hydroxymethyltransferase or C1-tetrahydrofolate synthase results in a rapid transfer of this bound folate to these enzymes. Evidence is also presented that the tightly bound folate is in equilibrium with solvent H4PteGlu5.

show abstract

Structural and functional characterization of a 5,10‐methenyltetrahydrofolate synthetase from Mycoplasma pneumoniae (GI: 13508087)

et al. 2005

View full text Add to dashboard Cite

Mycoplasma pneumoniae 5,10-methenyltetrahydrofolate synthetase [MTHFS; also known as 5-formyltetrahydrofolate cycloligase; Enzyme Commission (EC) 6.3.3.2] belongs to a large cycloligase protein family with 97 sequence homologues from bacteria to human. To help define the molecular (biochemical and biophysical) function of the M. pneumoniae MTHFS, we have previously determined its crystal structure at 2.2 A resolution (Chen et al., Proteins 2004;56:839-843). In this current study, activity assays confirmed the functionality of the recombinant protein, with K(m) = 165 microM for 5-formyltetrahydrofolate (5-FTHF) and K(m) = 166 microM for MgATP. The methenyltetrahydrofolate activity of M. pneumoniae MTHFS has a requirement for divalent metal ions with Mg2+ being most effective, and an absolute requirement for nucleoside 5'-triphosphates with adenosine triphosphate (ATP) being most effective. Crystallization in the presence of substrates (MgATP, with or without 5-FTHF) produced the complex structures of the protein with adenosine diphosphate (ADP) and phosphate at 2.2 A resolution; with ADP, phosphate, and 5-FTHF at 2.5 A resolution. These structures directly demonstrated that the role of Mg2+ in the reaction is to form the ATP--Mg2+-enzyme complex.

show abstract

Purification and properties of 5,10-methenyltetrahydrofolate synthetase from Lactobacillus casei.

Cited by 35 publications

References 23 publications

Hydrolysis of 5,10-Methenyltetrahydrofolate to 5-Formyltetrahydrofolate at pH 2.5 to 4.5

Hydrolysis of 5,10-Methenyltetrahydrofolate to 5-Formyltetrahydrofolate at pH 2.5 to 4.5

Properties of Tetrahydropteroylpentaglutamate Bound to 10-Formyltetrahydrofolate Dehydrogenase

Structural and functional characterization of a 5,10‐methenyltetrahydrofolate synthetase from Mycoplasma pneumoniae (GI: 13508087)

Contact Info

Product

Resources

About