Pyridoxine biosynthesis in yeast: participation of ribose 5-phosphate ketol-isomerase

To identify the genes involved in pyridoxine synthesis in yeast, auxotrophic mutants were prepared. After transformation with a yeast genomic library, a transformant (A22tl) was obtained from one of the auxotrophs. A22, which lost the pyridoxine auxotrophy. From an analysis of the plasmid harboured in A22tl the RKII gene coding for ribose 5-phosphate ketol-isomerase and residing on chromosome no. 15 was identified as the responsible gene. This notion was confirmed by gene disruption and tetrad analysis on a diploid prepared from the wild-type and the auxotroph. The site Of Mutation on the RKII gene was identified as position 566 with a transition from guanine to adenine, resulting in amino acid Substitution of Arg-189 with lysine. The enzymic activity of the Arg(189) --> Lys (R189K) Mutant of ribose 5-phosphate ketolisomerase was 0.6 % when compared with the wild-type enzyme. Loss of the structural integrity of the protein seems to be responsible for the greatly diminished activity, which eventually leads to a shortage of either ribose 5-phosphate or ribulose 5-phosphate as the starting or intermediary material for pyridoxine synthesis.