title: Purification and properties of 5,10-methenyltetrahydrofolate synthetase from Lactobacillus casei.
abstract: 5,10-Methenyltetrahydrofolate synthetase (EC 6.3.3.2), which catalyzes the ATP- and Mg2+ -dependent isomerization of 5-formyl- to 5,10-methenyltetrahydrofolate, has been purified 10,000-fold from Lactobacillus casei using sequential affinity chromatography on immobilized 5-formyltetrahydrofolate and ATP. The enzyme is homogeneous when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is monomeric with a molecular mass of 23,000 Da, and contains a high proportion of hydrophobic amino acids and a single cysteine residue. At 30 degrees C, the turnover number is 88 min-1, and the Km values at pH 6 for 5-formyltetrahydrofolate and Mg-ATP are 0.6 and 1.0 microM, respectively. The enzyme is specific for (6S)-5-formyltetrahydrofolate, but ATP can be replaced by other nucleoside 5'-triphosphates with varying efficiency. The purified enzyme is markedly stabilized by the non-ionic detergent, Tween 20.
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000838:Anions; D019731:Carbon-Nitrogen Ligases; D004355:Drug Stability; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D008025:Ligases / Q000302:isolation & purification* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D000838:Anions; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C021557:5,10-methenyltetrahydrofolate synthetase