Abstract

6,7-Dimethyl-8-ribityllumazine, the immediate biosynthetic precursor of riboflavin, is synthesized by condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate. The gene coding for 6,7-dimethyl-8-ribityllumazine synthase in Saccharomyces cerevisiae (RIB4) has been cloned by functional complementation of a mutant accumulating 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which can grow on riboflavin- or diacetyl- but not on 3,4-dihydroxy-2-butanone-supplemented media. Gene disruption of the chromosomal copy of RIB4 led to riboflavin auxotrophy and loss of enzyme activity. Nucleotide sequencing revealed a 169-base pair open reading frame encoding a 18.6-kDa protein. Hybridization analysis indicated that RIB4 is a single copy gene located on the left arm of chromosome XV. Overexpression of the RIB4 coding sequence in yeast cells under the control of the strong TEF1 promoter allowed ready purification of 6,7-dimethyl-8-ribityllumazine synthase to apparent homogeneity by a simple procedure. Initial structural characterization of 6,7-dimethyl-8-ribityllumazine synthase by gel filtration chromatography and both nondenaturing pore limit and SDS-polyacrylamide gel electrophoresis showed that the enzyme forms a pentamer of identical 16.8-kDa subunits. The derived amino acid sequence of RIB4 shows extensive homology to the sequences of the beta subunits of riboflavin synthase from Bacillus subtilis and other prokaryotes.

Highlights

  • Riboflavin, vitamin B2, is the precursor of flavin mononucleotide and flavin adenine dinucleotide, which function as coenzymes for a wide variety of enzymes in intermediate metabolism

  • Because the accumulated pyrimidine is the biosynthetic precursor of 6,7-dimethyl-8-ribityllumazine and both types of mutants are able to grow on media supplemented with riboflavin or diacetyl (diacetyl condenses nonenzymatically with 5-amino6-ribitylamino-2,4(1H,3H)-pyrimidinedione to yield 6,7-dimethyl-8-ribityllumazine), it was assumed that both types of mutants were affected in the synthesis of 6,7-dimethyl-8-ribityllumazine

  • In order to determine which specific metabolic step was affected in rib3 and rib4 mutants, we developed a nutritional test using chemically synthesized 3,4-dihydroxy2-butanone

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Summary

MOLECULAR CHARACTERIZATION OF THE GENE AND PURIFICATION OF THE ENCODED PROTEIN*

6,7-Dimethyl-8-ribityllumazine, the immediate biosynthetic precursor of riboflavin, is synthesized by condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate. The gene coding for 6,7-dimethyl-8-ribityllumazine synthase in Saccharomyces cerevisiae (RIB4) has been cloned by functional complementation of a mutant accumulating 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which can grow on riboflavin- or diacetylbut not on 3,4-dihydroxy-2-butanone-supplemented media. Whereas lower organisms are able to biosynthesize riboflavin, mammals have lost this capacity and, rely on its dietary ingestion to meet their metabolic needs As a consequence, this compound is commercially important as an additive in food industries, and several flavinogenic microorganisms, including some yeast species, are used in industry to produce riboflavin by fermentation [1]. The immediate precursor of riboflavin, 6,7-dimethyl-8-ribityllumazine (see Fig. 1, 4), is formed by condensation of 5-amino-6-ribitylamino-2,4(1H,3H)pyrimidinedione (see Fig. 1, 3) with 3,4-dihydroxy-2-butanone 4-phosphate (see Fig. 1, 2). We report here that rib mutants are defective in 6,7-dimethyl-8-ribityllumazine synthase

EXPERIMENTAL PROCEDURES
RESULTS
Purification factor
DISCUSSION
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