61] Purification and properties of 5,10-methylenetetrahydrofolate reductase from Clostridium formicoaceticum

Abstract

Publisher Summary This chapter describes the purification and properties of 5, 10-methylenetetrahydrofolate reductase from Clostridium formicoaceticum. It catalyzes the reduction of methylenetetrahydrofolate with reduced ferredoxin. The C. formicoaceticum methylenetetrahydrofolate reductase can be assayed by two different methods. One method, as described by Katzen and Buchanan is a sampling method which utilizes menadione as the electron carrier in the oxidation of [ methyl - 14 C]methyltetrahydrofolate to [ methylene - 14 C]methylenetetrahydrofolate. The [ methylene - 14 C]methylenetetrahydrofolate is in nonenzymatic equilibrium with tetrahydrofolate and H 14 CHO. The latter compound is trapped and extracted with 5,5-dimethyl-1,3-cyclohexanedione and toluene. The second assay is a continuous method which spectrophotometrically monitors the one-electron reduction of the artificial electron carrier, benzyl viologen (BV), at 555 nm. The reductase from C. formicoaceticum is oxygen labile and must be handled under anaerobic conditions. The assay for the reductase activity with benzyl viologen is conducted anaerobically to prevent reoxidation of reduced benzyl viologen by air. Assays have been performed in the physiological direction of the C. formicoaceticum reductase to determine the ability of the enzyme to transfer electrons from various reduced carriers to 5,10-methylenetetrahydrofolate.