Presence of Escherichia coli of a deaminase and a reductase involved in biosynthesis of riboflavin

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Abstract
Two enzymes were partially purified from extracts of E. coli B which together catalyze the conversion of the product of the action of GTP cyclohydrolase II, 2,5-diamino-6-oxy-4-(5''-phosphoribosylamino)pyrimidine, to 5-amino-2,6-dioxy-4-(5''-phosphoribitylamino)pyrimidine. These 2 compounds are currently thought to be intermediates in the biosynthesis of riboflavin. The enzymatic conversion occurs in 2 steps. The product of the action of GTP cyclohydrolase II first undergoes hydrolytic deamination at C 2 of the ring, followed by reduction of the ribosylamino group to a ribitylamino group. The enzyme which catalyzes the 1st step, herein called the deaminase, was purified 200-fold. The activity was assayed by detecting the conversion of the product of the reaction catalyzed by GTP cyclohydrolase II to a compound which reacts with butanedione to form 6,7-dimethyllumazine. The enzyme has a MW of about 80,000 and a pH optimum of 9.1. The dephosphorylated form of the substrate is not deaminated in the presence of the enzyme. The assay for the enzyme which catalyzes the 2nd step, referred to here as the reductase, involves the detection of the conversion of the product of the deaminase-catalyzed reaction to a compound which, after treatment with alkaline phosphatase, reacts with butanedione to form 6,7-dimethyl-8-ribityllumazine. The reductase has a MW of about 40,000 and a pH optimum of 7.5. Like the deaminase, the reductase does not act on the dephosphorylated form of its substrate. NADPH is required as a cofactor; NADH and the phosphate form can be used about 30%. The activity of neither enzyme is inhibited by riboflavin, FMN or FAD.