The Saccharomyces cerevisiae RIB4 Gene Codes for 6,7-Dimethyl- 8-ribityllumazine Synthase Involved in Riboflavin Biosynthesis

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 diacetylbut 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 ␤ subunits of riboflavin synthase from Bacillus subtilis and other prokaryotes.

Yeast Dihydroxybutanone Phosphate Synthase, an Enzyme of the Riboflavin Biosynthetic Pathway, Has a Second Unrelated Function in Expression of Mitochondrial Respiration

“…The rib3 null mutant used in this study shows an auxotrophic requirement of riboflavin for growth on glucose as reported previously (3). In contrast, the rib3 point mutant, like other pet mutants, grows on glucose in the absence of added riboflavin.…”

“…The mutant has been determined to have a point mutation in RIB3, a conserved gene of which the product, 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBP 1 synthase), catalyzes the first step in the riboflavin biosynthetic pathway (2). Complementation of aE280/U1 by RIB3 was unexpected in view of previous studies indicating that rib3 mutants require riboflavin for growth (3). The lack of correspondence in the phenotype of the rib3 point mutant and a mutant with a complete deletion of RIB3 prompted us to examine the biochemical properties of the mutant further.…”

Plant Riboflavin Biosynthesis

“…Sequence alignment of LS homologs. The amino acid sequence of the spinach LS precursor is aligned to the corresponding sequences from E. coli (8), B. subtilis (11), A. pleuropneumoniae (9), P. phosphoreum (10), and S. cerevisiae (12). Residues that are identical in at least four of the sequences are indicated by open squares, and those that are identical in all six sequences are indicated by filled black circles.…”

“…Section 1734 solely to indicate this fact. (12). In all cases, the subunit molecular mass of the encoded protein is small, ranging in size from ϳ16 to 17 kDa.…”

“…Since this strategy relies only on functional similarities between the disrupted host gene and the target gene of interest, it is ideally suited for cloning heterologous proteins that catalyze the same reaction. Indeed, several bacterial (18,19) and fungal (12,14) riboflavin biosynthetic enzymes, as well as plant GTP cyclohydrolase II (16), were identified through functional complementation of microbial riboflavin auxotrophs. Unexpectedly, higher plant LS is synthesized as a precursor protein with a cleavable N-terminal chloroplast targeting sequence.…”