Methylenetetrahydrofolate Dehydrogenase of the Amethopterin-resistant Strain Streptococcus faecium var. durans A k and Its Repressibility by Serine

Abstract

The methylenetetrahydrofolate dehydrogenase of the amethopterin-resistant strain Streptococcus faecium var. durans A k was purified 100-fold. Because it is extremely labile, this enzyme required protection by 1 m m nicotinamide adenine dinucleotide phosphate (NADP + ) during purification; 0.01 m m EADP + with 0.1% bovine plasma albumin stabilized the purified enzyme during storage at −20 C. Although the enzyme has properties of sulfhydryl enzymes, thiol compounds were not stabilizers. Oxidation of methylenetetrahydrofolate, catalyzed by the purified enzyme preparation, is NADP + -specific and yields methenyltetrahydrofolate and the reduced pyridine nucleotide. K m values for NADP + and for 5,10-methylenetetrahydrofolate (prepared as the formaldehyde adduct of biologically synthesized l , l -tetrahydrofolate) were calculated to be 0.021 and 0.026 m m , respectively. Neither purine bases and their derivatives nor serine inhibited the reaction. In growing cultures, the differential rate of synthesis of the methylenetetrahydrofolate dehydrogenase was dependent upon the composition of the medium. A medium which contained acid-hydrolyzed casein, and thus an exogenous source of serine, was repressive for this enzyme. In a serine-free, completely defined medium, the amount of folate added (for serine synthesis de novo) affected the duration of the initial exponential growth phase. At the termination of this phase, which primarily reflected the onset of a decreased rate of serine biosynthesis, synthesis of the methylenetetrahydrofolate dehydrogenase was derepressed. Exogenous serine in the completely defined medium prevented the derepression. Furthermore, physiological concentrations of l -serine were repressive not only for the dehydrogenase but also for the methenyltetrahydrofolate cyclohydrolase and the serine hydroxymethyl-transferase. Concomitantly, the differential rate of synthesis of the formyltetrahydrofolate synthetase of S. faecium var. durans A k was increased. Apparently, serine regulates the differential rates of syntheses of these enzymes.