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

Abstract

Two enzymes have been partially purified from extracts of Escherichia 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 5amino-2,6-dioxy-4-(5'-phosphoribitylamino)pyrimidine. These two compounds are currently thought to be intermediates in the biosynthesis of riboflavin. The enzymatic conversion occurs in two steps. The product of the action of GTP cyclohydrolase II first undergoes hydrolytic deamination at carbon 2 of the ring, followed by reduction of the ribosylamino group to a ribitylamino group. The enzyme which catalyzes the first step, herein called the "deaminase," has been 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 molecular weight of approximately 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 second 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 molecular weight of approximately 40,000 and a pH optimum of 7.5. Like the deaminase, the reductase does not act on the dephosphorylated form of its substrate. Reduced nicotinamide adenine dinucleotide phosphate is required as a cofactor; reduced nicotinamide adenine dinucleotide can be used about 30% as well as the phosphate form. The activity of neither enzyme is inhibited by riboflavin, FMN, or flavine adenine dinucleotide.

Biosynthesis of Vitamin B2 (Riboflavin)

“…The imidazole ring of GTP (structure 1 of Figure 1) is opened hydrolytically under release of formate accompanied by release of pyrophosphate, which is catalyzed by GTP cyclohydrolase II (12,13,16,26,41,42,58,83,108,109). The enzyme product 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5 -phosphate (2 in Figure 1) is converted to 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5 -phosphate (5 in Figure 1) by two reaction steps, involving the hydrolytic cleavage of the position 2 amino group of the heterocyclic ring and the reduction of the ribosyl side chain affording the ribityl side chain of the vitamin (33,83). The sequence of these reaction steps varies in different organisms.…”

“…The sequence of these reaction steps varies in different organisms. In eubacteria, the deamination precedes the side chain reduction (33). In yeasts and fungi, the reduction precedes the deamination (10,54,64,103).…”

“…Both enzyme activities require Mg 2+ . The reductase requires NADPH or NADH as cofactor (33,52). Genes specifying similar putative proteins have been found in a variety of microorganisms.…”

Biosynthesis of vitamin B2 in plants

“…The conversion of 2,5‐diamino‐6‐ribosylamino‐4(3 H )‐pyrimidinone 5′‐phosphate ( 2 ) into 5‐amino‐6‐ribitylamino‐2,4(1 H ,3 H )‐pyrimidinedione 5′‐phosphate ( 5 ) had been shown earlier to proceed via different intermediates in fungi and bacteria (Bacher and Lingens, 1970; Burrows and Brown, 1978; Nielsen and Bacher, 1981). In yeasts, the reaction sequence is initiated by the reductive conversion of the ribosyl side chain of 2 into the ribityl side chain of 2,5‐diamino‐6‐ribitylamino‐4(3 H )‐pyrimidinedione 5′‐phosphate ( 4 ); subsequent deamination of the pyrimidine moiety yields 5 .…”

Cloning, Expression, Purification, Characterization, Crystallization and X-Ray Diffraction of Bifunctional Pyrimidine Deaminase/Reductase from Shigella flexneri 2a

“…The production of riboflavin from GTP is catalyzed by the enzymes GTP cyclohydrolase II, riboflavin deaminase/reductase, 3, 4dihydroxy-2-butanone-4-phosphate synthase, lumazine synthase and riboflavin synthase [1]. The deamination and reduction steps have been shown to proceed in the opposite order in yeast and Escherichia coli [2,3]. Most eubacteria contain a bifunctional protein, for instance Bacillus subtilis RibD (BsRibD) is composed of an N-terminal deaminase domain (D domain) and a C-terminal reductase domain (R domain) [4].…”