Keiko Tazuya

Origin of the nitrogen atom of pyridoxine in Saccharomyces cerevisiae.

The origin of the nitrogen atom of pyridoxine was studied in Saccharomyces cerevisiae. The 15N atom of 15NH4Cl added to the growth medium as the nitrogen source was incorporated efficiently into the nitrogen atom of pyridoxine. The competitive effects of 14N-amino acids on the incorporation of 15NH4Cl were examined. Incorporation of 15N into pyridoxine was inhibited by glutamine. The label of L-[amide-15N]glutamine was incorporated effectively into pyridoxine in S. cerevisiae. On the other hand, the label from L-[amide-15N]glutamine was not incorporated into pyridoxine in Escherichia coli. These findings suggest that the biosynthetic pathway of pyridoxine in S. cerevisiae differs from that in E. coli.

Incorporation of histidine into the pyrimidine moiety of thiamin in Saccharomyces cerevisiae

To investigate the incorporation of histidine into the pyrimidine moiety of thiamin in eukaryotes, Saccharomyces cerevisiae was grown in a synthetic medium in the presence of 15N- or 14C-labeled histidine. Two 15N-atoms of DL-[1,3-15N2]histidine were incorporated into the N-3 and amino-N atom at C-4 of pyrimidine. Furthermore, incorporation of the 15N-amino group of aspartate, the origin of the N-1 of histidine, into the N-3 of pyrimidine shows that N-3 and the amino-N atom at C-4 of pyrimidine are derived from N-1 and N-3 of histidine, respectively. In contrast, the 15N atom of DL-[amino-15N]histidine was not incorporated into the molecule, whereas L-[2-14C]histidine was incorporated directly into the pyrimidine. We conclude that N-1, C-2, and N-3 of histidine are the origins of the N-3, C-4, and amino-N at C-4 of the pyrimidine in thiamin synthesized by S. cerevisiae.

The origin of the sulfur atom of thiamin.

The incorporation of the sulfur atom of 35S-labeled amino acids into thiamin in Escherichia coli and Saccharomyces cerevisiae was studied. The specific radioactivity of the S atoms was incorporated at similar levels into thiamin and cysteine residues in cell proteins. However, the specific radioactivity of the S atoms from [35S]methionine was not incorporated into thiamin but into methionine residues in cell proteins. Thus, the origin of the S atom of thiamin was established as being the S atom of cysteine. No activity from [U-14C]cysteine was recovered in thiamin, proving that the carbon skeleton of this amino acid was not utilized in synthesizing the thiazole moiety of thiamin.

Enzymatic and structural characterization of an archaeal thiamin phosphate synthase.

Studies on thiamin biosynthesis have so far been achieved in eubacteria, yeast and plants, in which the thiamin structure is formed as thiamin phosphate from a thiazole and a pyrimidine moiety. This condensation reaction is catalyzed by thiamin phosphate synthase, which is encoded by the thiE gene or its orthologs. On the other hand, most archaea do not seem to have the thiE gene, but instead their thiD gene, coding for a 2-methyl-4-amino-5-hydroxymethylpyrimidine (HMP) kinase/HMP phosphate kinase, possesses an additional C-terminal domain designated thiN. These two proteins, ThiE and ThiN, do not share sequence similarity. In this study, using recombinant protein from the hyperthermophile archaea Pyrobaculum calidifontis, we demonstrated that the ThiN protein is an analog of the ThiE protein, catalyzing the formation of thiamin phosphate with the release of inorganic pyrophosphate from HMP pyrophosphate and 4-methyl-5-β-hydroxyethylthiazole phosphate (HET-P). In addition, we found that the ThiN protein can liberate an inorganic pyrophosphate from HMP pyrophosphate in the absence of HET-P. A structure model of the enzyme-product complex of P. calidifontis ThiN domain was proposed on the basis of the known three-dimensional structure of the ortholog of Pyrococcus furiosus. The significance of Arg320 and His341 residues for thiN-coded thiamin phosphate synthase activity was confirmed by site-directed mutagenesis. This is the first report of the experimental analysis of an archaeal thiamin synthesis enzyme.

Isotopically labeled precursors and mass spectrometry in elucidating biosynthesis of pyrimidine moiety of thiamin in Saccharomyces cerevisiae.

Publisher Summary This chapter investigates pyrimidine biosynthesis in Saccharomyces cerevisiae ( S. cerevisiae )by gas chromatography–mass spectrometry (GC–MS) analysis, using a stable isotope-labeled tracer. The amide nitrogen atom of glutamine is incorporated into N-3 and amino nitrogen (amino-N) at C-4 of the pyrimidine moiety of thiamin in S. cerevisiae . However, addition of casamino acids to the medium decreases incorporation of the amide nitrogen atom of glutamine into the pyrimidine. This suggests that another amino acid in casamino acids is the direct precursor. To determine the direct precursor, the competitive effects of 14 N-labeled amino acids on the incorporation of 15 NH 4 Cl into pyrimidine are investigated. To confirm the direct incorporation of histidine, S. cerevisiae is grown in the presence of L -[1,3- 15 N 2 ]histidine. The two nitrogen atoms of the imidazole ring in histidine are incorporated directly into pyrimidine. The base peak m / z 122 is shifted to m / z 124. The shift of the fragment ion at m / z 54 to 55 shows that one of the nitrogen atoms of imidazole in histidine is incorporated into the amino-N of pyrimidine. The fragment ion at m / z 81 contains the N-1 and amino-N atoms of pyrimidine. The one nitrogen of histidine is incorporated into this fragment. These results show that the nitrogen atom of histidine is incorporated into the amino-N atom but not the N-1 of pyrimidine. Therefore, the other nitrogen of histidine is incorporated into the N-3 of pyrimidine.