Involvement of thiaminase II encoded by the THI20 gene in thiamin salvage of Saccharomyces cerevisiae.
The physiological significance of thiaminase II, which catalyzes the hydrolysis of thiamin, has remained elusive for several decades. The C-terminal domains of THI20 family proteins (THI20/21/22) and the whole region of PET18 gene product of Saccharomyces cerevisiae are homologous to bacterial thiaminase II. On the other hand, the N-terminal domains of THI20 and THI21 encode 2-methyl-4-amino-5-hydroxymethylpyrimidine kinase and 2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate kinase involved in the thiamin synthetic pathway. In this study, it was first indicated that the C-terminal domains of the THI20 family and PET18 are not required for de novo thiamin synthesis in S. cerevisiae, using a quadruple deletion strain expressing the N-terminal domain of THI20. Biochemical analysis using cell-free extracts and recombinant proteins demonstrated that yeast thiaminase II activity is exclusively encoded by THI20. It appeared that Thi20p has an affinity for the pyrimidine moiety of thiamin, and HMP produced by the thiaminase II activity is immediately phosphorylated. Thi20p was found to participate in the formation of thiamin from two synthetic antagonists, pyrithiamin and oxythiamin, by hydrolyzing both antagonists and phosphorylating HMP to give HMP pyrophosphate. Furthermore, 2-methyl-4-amino-5-aminomethylpyrimidine, a presumed naturally occurring thiamin precursor, was effectively converted to HMP by incubation with Thi20p. It is proposed that the thiaminase II activity of Thi20p is involved in the thiamin salvage pathway by catalyzing the hydrolysis of HMP precursors in S. cerevisiae.
Onozuka
Mari
M
Department of Radioisotope Laboratory, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Konno
Hiroyuki
H
Kawasaki
Yuko
Y
Akaji
Kenichi
K
Nosaka
Kazuto
K
D017353:Gene Deletion; D006867:Hydrolases / Q000378:metabolism*; D010119:Oxythiamine / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000235:genetics / Q000378:metabolism; D011743:Pyrimidines / Q000378:metabolism; D011745:Pyrithiamine / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013831:Thiamine / Q000378:metabolism*
C528483:PET18 protein, S cerevisiae; D011743:Pyrimidines; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D010119:Oxythiamine; D011745:Pyrithiamine; C013116:2-methyl-4-amino-5-aminomethylpyrimidine; D017853:Phosphotransferases (Alcohol Group Acceptor); C059599:hydroxymethylpyrimidine kinase; C108436:4-amino-5-(hydroxymethyl)-2-methylpyrimidine phosphate kinase; D017856:Phosphotransferases (Phosphate Group Acceptor); C528484:THI22 protein, S cerevisiae; D006867:Hydrolases; C022731:thiaminase II; C012068:toxopyrimidine; C030986:pyrimidine; D013831:Thiamine