Abstract
The enzymes concerned with thiamin phosphates metabolism in Saccharomyces cerevisae were characterized by genetic and biological analyses. There exist two species of acid phosphatase in yeast periplasmic space ; one, repressible by Pi and the other, repressible by thiamin. Thiamin-repressible acid phosphatase encoded by PHO3 gene was ascertained to physiologically catalyze the hydrolysis of extracellular thiamin phosphates, thus participating in utilization of the thiamin moiety of the phosphates. Next, the THI80 gene encoding for a thiamin pyrophosphokinase and the THI6 gene encoding for a bifunctional thiamin-phosphate pyrophosphorylase/hydroxyethylthiazole kinase were isolated from yeast genomic library by functional complementation of thi80 and thi6 mutant, respectively. The THI80 gene contained a 957-bp open reading frame coding a 319-aa polypeptide (Mw : 36, 616), and the THI80 gene was proved to be essential for growth by a gene disruption study. The THI6 gene contained a 1,620-bp open reading frame coding a 540-aa polypeptide (Mw : 58, 058), and the functional domains of the two enzvme activities in the THI6 gene were determined to locate independently by a in vitro mutagenesis. Northern blot analysis demonstrated that PHO3, THI80 and THI6 gene expression are regulated at the mRNA level by intracellular thiamin pyrophosphate, and they require the positive regulatory factors encoded by the THI2 and THI3 genes in S. cerevisiae.
