Nosaka K, Nishimura H, Kawasaki Y, Tsujihara T, Iwashima A
Department of Biochemistry, Kyoto Prefectural University of Medicine, Japan.
J Biol Chem. 1994 Dec 2;269(48):30510-6.
Thiamin-phosphate pyrophosphorylase (TMP-PPase; EC 2.5.1.3) involved in de novo synthesis of thiamin in Saccharomyces cerevisiae is a bifunctional enzyme with 4-methyl-5-beta-hydroxyethylthiazole kinase (Th-kinase; EC 2.7.1.50) activity, which is an octamer of identical 60-kDa subunits (Kawasaki, Y. (1993) J. Bacteriol. 175, 5153-5158). Previous study demonstrated that the activities of both TMP-PPase and Th-kinase are reduced by the mutation of a single nuclear gene, designated THI6. We have cloned the THI6 gene from a yeast genomic library by functional complementation of the thi6 mutant and determined by DNA blot analysis that THI6 is located on chromosome XVI. The nucleotide sequence of the THI6 gene contained an open reading frame of 1,620 base pairs encoding a 540-amino acid polypeptide with a calculated molecular weight of 58,058, which is similar to the determined molecular mass of the purified bifunctional enzyme. Gene disruption demonstrated that the thi6 null strain is auxotrophic for thiamin, indicating that the THI6 protein is essential for thiamin synthesis in yeast. A recently isolated thi6 mutant, thi6-3, bearing a replacement of Glu370 by Lys370, showed a decrease in only Th-kinase activity, proving that the THI6 gene of S. cerevisiae encodes a structural gene of the thiamin biosynthetic bifunctional enzyme. Furthermore, complementation analysis of the thi6 null strain with the modified THI6 DNAs by a 12-nucleotide linker insertion suggested that a region from amino acids 138 to 187 and that from amino acids 370 to 453 are involved in functional domains of TMP-PPase and Th-kinase, respectively, whereas the COOH-terminal region is necessary for both enzyme activities. Strains conferring no Th-kinase but slight TMP-PPase activity could grow in medium without thiamin, suggesting that 4-methyl-5-beta-hydroxyethylthiazole is not involved in the pathway of de novo synthesis of thiamin via 4-methyl-5-beta-hydroxyethylthiazole monophosphate. Northern blot analysis demonstrated that THI6 gene expression is regulated at the mRNA level by intracellular thiamin pyrophosphate, a coenzyme form of thiamin, and that it requires the positive regulatory factors encoded by the THI2 and THI3 genes.
硫胺素磷酸焦磷酸化酶（TMP-PPase；EC 2.5.1.3）参与酿酒酵母中硫胺素的从头合成，是一种具有4-甲基-5-β-羟乙基噻唑激酶（Th-激酶；EC 2.7.1.50）活性的双功能酶，它是由相同的60 kDa亚基组成的八聚体（川崎，Y.（1993年）《细菌学杂志》175，5153 - 5158）。先前的研究表明，单个核基因THI6的突变会降低TMP-PPase和Th-激酶的活性。我们通过thi6突变体的功能互补从酵母基因组文库中克隆了THI6基因，并通过DNA印迹分析确定THI6位于第十六条染色体上。THI6基因的核苷酸序列包含一个1620个碱基对的开放阅读框，编码一个540个氨基酸的多肽，计算分子量为58,058，这与纯化的双功能酶的测定分子量相似。基因破坏表明thi6缺失菌株对硫胺素营养缺陷，表明THI6蛋白对酵母中的硫胺素合成至关重要。最近分离的thi6突变体thi6 - 3，其第370位谷氨酸被赖氨酸取代，仅Th-激酶活性降低，证明酿酒酵母的THI6基因编码硫胺素生物合成双功能酶的结构基因。此外，通过12个核苷酸的接头插入对thi6缺失菌株与修饰的THI6 DNA进行互补分析表明，氨基酸138至187区域和氨基酸370至453区域分别参与TMP-PPase和Th-激酶的功能结构域，而COOH末端区域对两种酶活性都是必需的。不具有Th-激酶但具有轻微TMP-PPase活性的菌株可以在不含硫胺素的培养基中生长，这表明4-甲基-5-β-羟乙基噻唑不参与通过4-甲基-5-β-羟乙基噻唑单磷酸的硫胺素从头合成途径。Northern印迹分析表明，THI6基因表达在mRNA水平上受硫胺素焦磷酸（硫胺素的辅酶形式）的调节，并且它需要由THI2和THI3基因编码的正调控因子。
