ISOLATION AND CHARACTERIZATION OF A THIAMIN PYROPHOSPHOKINASE GENE, THI80, FROM SACCHAROMYCES-CEREVISIAE

The thi80 mutant of Saccharomyces cerevisiae (Nishimura, H., Kawasaki, Y., Nosaka, K., Kaneko, Y., and Iwashima, A. (1991) J. Bacteriol. 173 , 2716-2719) shows markedly reduced activity of thiamin pyrophosphokin ase (TPK; EC 2.7.6.2). We have isolated a DNA fragment carrying the TH I80 gene from a yeast genomic library by its ability to complement con stitutive synthesis of the thiamin-repressible acid phosphatase, encod ed by the PHO3 gene, of thi80 mutant cells. On the other hand, the thi 80 locus was found to be located 3.3 centimorgans proximal to the smp3 locus on the right arm of chromosome XV by genetic mapping analysis, and one more fragment bearing the THI80 gene trailing SMP3 gene was ob tained by the plasmid eviction method. The nucleotide sequence of the overlapped region between the two isolated DNAs contained an open read ing frame of 957 base pairs, encoding a 319-amino acid polypeptide wit h a calculated molecular weight of 36,616. When the intact THI80 open reading frame was expressed as a fusion protein carrying three vector- encoded amino acids at its N terminus in Escherichia coli lacking TPK, marked TPK activity was detected in the procaryotic cells, proving th at the THI80 gene of S. cerevisiae encodes a structural gene of TPK. A gene disruption experiment demonstrated that the THI80 gene was essen tial for growth, and therefore, revealed that TPK is the only enzyme c apable of synthesizing thiamin pyrophosphate in yeast. Studies of Nort hern blot analysis and the enzyme assay demonstrated that the THI80 ge ne expression is regulated mainly at the mRNA level by the intracellul ar thiamin pyrophosphate and requires the positive regulatory factors encoded by THI2 and THI3 genes. However, unlike thiamin-repressible ac id phosphatase and the enzymes involved in thiamin synthesis of S. cer evisiae, TPK was found to be expressed constitutively at a low level a nd incompletely repressed by exogenous thiamin.