THE SACCHAROMYCES-CEREVISIAE RIB4 GENE CODES FOR 6,7-DIMETHYL-8-RIBITYLLUMAZINE SYNTHASE INVOLVED IN RIBOFLAVIN BIOSYNTHESIS - MOLECULAR CHARACTERIZATION OF THE GENE AND PURIFICATION OF THE ENCODED PROTEIN

6,7-Dimethyl-8-ribityllumazine, the immediate biosynthetic precursor o f riboflavin, is synthesized by con densation of 5 amino 6-ribitylamin o-2,4(1H,3H)-pyrimidinedione with 3,4-dihydroxy-2-butanone 4 phosphate . The gene coding for 6,7-dimethyl-8-ribityllumazine synthase in Sacch aromyces cerevisiae (RIB4) has been cloned by functional complementati on of a mutant accumulating 5-amino-6-ribitylamino-2,4( 1H, 3H) -pyrim idinedione, which can grow on riboflavin- or diacetyl-but not on 3,4-d ihydroxy-2-butanone-supplemented media, Gene disruption of the chromos omal copy of RIB4 led to riboflavin auxotrophy and loss of enzyme acti vity, Nucleotide sequencing revealed a 169-base pair open reading fram e encoding a 18.6-kDa protein. Hybridization analysis indicated that R IB4 is a single copy gene located on the left arm of chromosome XV. Ov erexpression of the RIB4 coding sequence in yeast cells under the cont rol of the strong TEF1 promoter allowed ready purification of 6,7-dime thyl-8-ribityllumazine synthase to apparent homogeneity by a simple pr ocedure. Initial structural characterization of 6,7-dimethyl-8-ribityl lumazine synthase by gel filtration chromatography and both nondenatur ing pore limit and SDS-polyacrylamide gel electrophoresis showed that the enzyme forms a pentamer of identical 16.8-kDa subunits. The derive d amino acid sequence of RIB4 shows extensive homology to the sequence s of the beta subunits of riboflavin synthase from Bacillus subtilis a nd other prokaryotes.