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

J Biol Chem. 1995 Oct 6;270(40):23801-7. doi: 10.1074/jbc.270.40.23801.

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacteria / enzymology
  • Bacteria / genetics
  • Base Sequence
  • Chromosome Mapping
  • Cloning, Molecular
  • DNA, Fungal / genetics
  • Genes, Fungal*
  • Genetic Complementation Test
  • Molecular Sequence Data
  • Multienzyme Complexes / genetics*
  • Multienzyme Complexes / isolation & purification
  • Multienzyme Complexes / metabolism*
  • Mutation
  • Open Reading Frames
  • Protein Conformation
  • Pteridines / metabolism
  • Riboflavin / biosynthesis*
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics*
  • Sequence Homology, Amino Acid
  • Species Specificity
  • Transformation, Genetic

Substances

  • DNA, Fungal
  • Multienzyme Complexes
  • Pteridines
  • 6,7-dimethyl-8-ribityllumazine
  • 6,7-dimethyl-8-ribityllumazine synthase
  • Riboflavin

Associated data

  • GENBANK/Z21620