CLONING AND CHARACTERIZATION OF THE FIRST 2 GENES OF THE NONOXIDATIVEPART OF THE SACCHAROMYCES-CEREVISIAE PENTOSE-PHOSPHATE PATHWAY

We have cloned and characterized the two remaining unknown genes of th e non-oxidative part of the pentose-phosphate pathway of Saccharomyces cerevisiae encoding the enzymes D-ribulose-5-phosphate 3-epimerase (R pe1p) and D-ribose-5-phosphate ketol-isomerase (Rki1p). Rpe1p has an u nexpected high specific activity of 2148 mUx(mg protein)(-1) in crude extracts. Deletion mutants of RPE1 show no enzyme activity and are una ble to grow on D-xylulose. Unexpectedly haploid rki1 deletion mutants are not viable. Functional expression of RKI1 was demonstrated followi ng an increase of gene dosage in the haploid rki1 deletion mutant, whi ch restored viability and specific D-ribose-5-phosphate ketol-isomeras e activity. Both enzymes show high similarity to the deduced protein s equences of various open reading frames, expressed sequence tags or cD NAs from different organisms.