Nosaka K, Kaneko Y, Nishimura H, Iwashima A
Department of Biochemistry, Kyoto Prefectural University of Medicine, Japan.
J Biol Chem. 1993 Aug 15;268(23):17440-7.
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 pyrophosphokinase (TPK; EC 2.7.6.2). We have isolated a DNA fragment carrying the THI80 gene from a yeast genomic library by its ability to complement constitutive synthesis of the thiamin-repressible acid phosphatase, encoded by the PHO3 gene, of thi80 mutant cells. On the other hand, the thi80 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 obtained by the plasmid eviction method. The nucleotide sequence of the overlapped region between the two isolated DNAs contained an open reading frame of 957 base pairs, encoding a 319-amino acid polypeptide with 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 that the THI80 gene of S. cerevisiae encodes a structural gene of TPK. A gene disruption experiment demonstrated that the THI80 gene was essential for growth, and therefore, revealed that TPK is the only enzyme capable of synthesizing thiamin pyrophosphate in yeast. Studies of Northern blot analysis and the enzyme assay demonstrated that the THI80 gene expression is regulated mainly at the mRNA level by the intracellular thiamin pyrophosphate and requires the positive regulatory factors encoded by THI2 and THI3 genes. However, unlike thiamin-repressible acid phosphatase and the enzymes involved in thiamin synthesis of S. cerevisiae, TPK was found to be expressed constitutively at a low level and incompletely repressed by exogenous thiamin.
酿酒酵母的thi80突变体（西村浩、川崎洋、野坂克、金子洋、岩岛昭（1991年）《细菌学杂志》173卷，2716 - 2719页）显示硫胺素焦磷酸激酶（TPK；EC 2.7.6.2）的活性显著降低。我们通过其对thi80突变体细胞中由PHO3基因编码的硫胺素可阻遏酸性磷酸酶组成型合成的互补能力，从酵母基因组文库中分离出了携带THI80基因的DNA片段。另一方面，通过遗传图谱分析发现thi80位点位于第十五号染色体右臂上smp3位点近端3.3厘摩处，并且通过质粒排除法获得了另一个携带尾随SMP3基因的THI80基因的片段。两个分离出的DNA之间重叠区域的核苷酸序列包含一个957个碱基对的开放阅读框，编码一个319个氨基酸的多肽，计算分子量为36,616。当完整的THI80开放阅读框在缺乏TPK的大肠杆菌中作为在其N端携带三个载体编码氨基酸的融合蛋白表达时，在原核细胞中检测到了显著的TPK活性，证明酿酒酵母的THI80基因编码TPK的一个结构基因。基因破坏实验表明THI80基因对生长至关重要，因此揭示了TPK是酵母中唯一能够合成硫胺素焦磷酸的酶。Northern印迹分析和酶活性测定研究表明，THI80基因表达主要在mRNA水平受细胞内硫胺素焦磷酸调控，并且需要THI2和THI3基因编码的正调控因子。然而，与酿酒酵母的硫胺素可阻遏酸性磷酸酶和参与硫胺素合成的酶不同，发现TPK以低水平组成型表达，并且不受外源硫胺完全阻遏。
