ISOLATION AND CHARACTERIZATION OF THE THI6 GENE ENCODING A BIFUNCTIONAL THIAMIN-PHOSPHATE PYROPHOSPHORYLASE HYDROXYETHYLTHIAZOLE KINASE FROM SACCHAROMYCES-CEREVISIAE

Thiamin-phosphate pyrophosphorylase (TMP-PPase; EC 2.5.1.3) involved i n de novo synthesis of thiamin in Saccharomyces cerevisiae is a bifunc tional enzyme with 4-methyl-5-beta-hydroxyethylthiazole kinase (Th-kin ase; EC 2.7.1.50) activity, which is an octamer of identical 60-kDa su bunits (Kawasaki, Y. (1993) J. Bacteriol. 175, 5153-5158). Previous st udy demonstrated that the activities of both TMP PPase and Th-kinase a re reduced by the mutation of a single nuclear gene, designated THI6. We have cloned the THI6 gene from a yeast genomic library by functiona l complementation of the this mutant and determined by DNA blot analys is that THI6 is located on chromosome XVI. The nucleotide sequence of the THI6 gene contained an open reading frame of 1,620 base pairs enco ding a 540 amino acid polypeptide with a calculated molecular weight o f 58,058, which is similar to the determined molecular mass of the pur ified bifunctional enzyme. Gene disruption demonstrated that the this null strain is auxotrophic for thiamin, indicating that the THI6 prote in is essential for thiamin synthesis in yeast, A recently isolated th is mutant, thi6-3, bearing a replacement of Glu(370) by Lys(370), show ed a decrease in only Th-kinase activity, proving that the THI6 gene o f S. cerevisiae encodes a structural gene of the thiamin biosynthetic bifunctional enzyme. Furthermore, complementation analysis of the this null strain with the modified THI6 DNAs by a 12-nucleotide linker ins ertion suggested that a region from amino acids 138 to 187 and that fr om amino acids 370 to 453 are involved in functional domains of TMP PP ase and Th-kinase, respectively, whereas the COOH-terminal region is n ecessary for both enzyme activities. Strains conferring no Th-kinase b ut slight TMP-PPase activity could grow in medium without thiamin, sug gesting that 4-methyl-beta-hydroxyethylthiazole is not involved in the pathway of de novo synthesis of thiamin via 4-methyl-5-beta-hydroxyet hylthiazole monophosphate. Northern blot analysis demonstrated that TH I6 gene expression is regulated at the mRNA level by intracellular thi amin pyrophosphate, a coenzyme form of thiamin, and that it requires t he positive regulatory factors encoded by the THI2 and THI3 genes.