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 (A22t1) was obtained from one of the auxotrophs, A22, which lost the pyridoxine auxotrophy. From an analysis of the plasmid harboured in A22t1, the RKI1 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 RKI1 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 Arg189-->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.
Kondo
Hiroki
H
Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Kawazu 680-4, Iizuka 820-8502, Japan. kondo@bse.kyutech.ac.jp
Nakamura
Yoriko
Y
Dong
Yi-Xin
YX
Nikawa
Jun-ichi
J
Sueda
Shinji
S
D019747:Aldose-Ketose Isomerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D002352:Carrier Proteins; D002874:Chromosome Mapping; D005800:Genes, Fungal; D020125:Mutation, Missense; D017354:Point Mutation; D011736:Pyridoxine / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012267:Ribosemonophosphates / Q000378:metabolism; D012274:Ribulosephosphates / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D011993:Recombinant Fusion Proteins; D012267:Ribosemonophosphates; D012274:Ribulosephosphates; D029701:Saccharomyces cerevisiae Proteins; C117606:VPS54 protein, S cerevisiae; D033921:Vesicular Transport Proteins; C031524:ribulose 5-phosphate; C031626:ribose-5-phosphate; D019747:Aldose-Ketose Isomerases; C487718:RKI1 protein, S cerevisiae; C022548:ribosephosphate isomerase; D011736:Pyridoxine