Naturally Occurring Forms of Folic Acid

Previously (1, 6), it was shown that prefolic A, in crude form, was converted to N5-formyltetrahydrofolic acid by two separate enzymatic reactions. First, in the presence of a suitable electron acceptor, prefolic A is converted to tetrahydrofolic acid by a flavin adenine dinucleotide-linked enzyme system. Then, in the presence of transformylase and formyl-L-glutamate, tetrahydrofolic acid is formylated to N5-formyltetrahydrofolic acid. The conversion of prefolic A to tetrahydrofolic acid was postulated to be an oxidative reaction, mediated through a flavin-linked enzyme system with the dye as the electron acceptor. More recently, it has been shown that formaldehyde is one of the products formed during the oxidation of prefolic A to tetrahydrofolic acid (7). The experiments leading to this observation were prompted by the reported synthesis of a new folic acid intermediate in methionine biosynthesis by Larrabee and Buchanan (8). These investigators showed that this folic acid derivative contained 1 mole of N-methyl group per mole of compound. During the discussion which followed the presentation of “Interrelationship of Prefolic A and Tetrahydrofolic Acid” (9), Buchanan suggested the possibility that prefolic A and the newly discovered intermediate of methionine biosynthesis might be the same compound. In the present investigation, results have been obtained with purified natural prefolic A, and in some experiments with chemically synthesized2 prefolic A (lo), which confirm those obtained with the crude material (1). Also, it will be shown that menadione is a more effective electron acceptor than the dyes used previously in the oxidation of prefolic A to tetrahydrofolic acid, and that for every mole of tetrahydrofolic acid formed from prefolic A, 1 mole of formaldehyde is released. In addition, evidence will be presented for the enzymatic synthesis of prefolic ,4 from tetrahydrofolic acid and formaldehyde (or methanol) in the presence of flavin adenine dinucleotide and reduced diphosphopyridine nucleotide.