The Biosynthesis of Folic Acid I. SUBSTRATE AND COFACTOR REQUIREMENTS FOR ENZYMATIC SYNTHESIS BY CELL-FREE EXTRACTS OF ESCHERICHIA COLI*

The formation of folic acid compounds from pteridines and p-aminobenzoic acid by whole bacterial cells has been studied by Korte et al. (1, 2), who reported that C%xanthopterin (2-amino4,6-dihydroxypteridine) could be incorporated into folic acid by several bacterial species, and by Weygand et al. (3), who found that certain strains of bacteria can be adapted to utilize 2.amino-4-hydroxy-6-formylpteridine for folate production. The initial experiments concerned with folate synthesis in cellfree extracts were done by Katunuma.1 His evidence indicated that extracts of Mycobacterium awium could convert either xanthopterin or 2-amino-4-hydroxy-6-carboxypteridine and paminobenzoylglutamic acid to folate. Evidence was also presented to show that these extracts were able to catalyze the formation of p-aminobenzoylglutamic acid from p-aminobenzoic acid and glutamate with p-aminobenzoic acid-adenylate as an intermediate in the system. Somewhat later, Shiota (4) found that extracts of Lactobacillus arabinosus contained enzymes which, in the presence of adenosine triphosphate, could utilize as substrate either 2-amino-4-hydroxy-6-hydroxymethylpteridine or 2-amino-4-hydroxy-6-formylpteridine (or the corresponding tetrahydro forms of these compounds) for the synthesis of either pteroic acid (from p-aminobenzoic acid) or folic acid (from paminobenzoylglutamic acid). Brown (5) found that extracts of Escherichia coli contained a similar enzyme system for the synthesis of either pteroate or folate. The E. coli system could utilize as substrate only 2-amino-4-hydroxy-6-formylpteridine or reduced forms of this compound and 2-amino-4-hydroxy-6hydroxymethylpteridine (6, 7) ; reduced diphosphopyridine nucleotide and flavin adenine nucleotide, as well as adenosine triphosphate, also were necessary components of the reaction mixtures (7). Because p-aminobenzoic acid was a much more effective substrate for pteroate synthesis than was p-aminobenzoylglutamic acid for folate synthesis, it was postulated that pteroic acid (or a reduced form of pteroate) is an intermediate in the biosynthesis of folate in E. coli (6,7). The reduced forms of pteridines appeared to be used more effectively as substrate than