Saccharomyces cerevisiae TPP 2-oxo acid decarboxylases

The irreversible decarboxylation step, which commits 2-oxo acids to the Ehrlich pathway, was initially attributed to pyruvate decarboxylase. However, the yeast genome was shown to harbour no fewer than 5 genes that show sequence similarity with thiamine-diphosphate dependent decarboxylase genes. Three of these (PDC1, PDC5 and PDC6) encode pyruvate decarboxylases { while ARO10 and THI3 represent alternative candidates for Ehrlich-pathway decarboxylases. Transcriptome analysis and decarboxylase activity measurements on an S. cerevisiae aro10 strain, a double aro10 thi3 deletion strain and a quadruple pdc1,5,6,aro10 mutant strains grown in carbon–limited chemostat with phenylalanine as nitrogen source indicated that: i) PDC5 is strongly upregulated in an aro10 background (Fig. 2) and also encodes a broad-substrate α-keto acid decarboxylase. ii) PDC5 expression depends on the presence of THI3 (Fig. 2), and iii) in contrast to cell extracts from a strain expressing ARO10 only (pdc1,5,6, thi3), cell extract from a strain that only contains THI3 (pdc1,5,6,aro10) did not produce any α-keto acid decarboxylase activity . THI3 has recently been demonstrated to be involved in regulation of thiamine homeostasis in S. cerevisiae, which further suggests that its role in the Ehrlich pathway may be regulatory rather than catalytic. A systematic investigation of the catalytic properties of all five (putative) TPP-dependent decarboxylases (Aro10p, Thi3p, Pdc1p, Pdc5p, Pdc6p) is essential for a final resolution of the substrate specificity of these key enzymes in the Ehrlich pathway. Keywords: Strain comparison Comparison of glucose limited chemostat cultivations with phenylalanine as sole nitrogen sources of the following strains: - CENPK113-7D MATa MAL2-8c SUC2 - CENPK555-4A MATa MAL2-8c SUC2 ydr380w delta - CENPK5632-3B MATalpha MAL2-8c SUC2 ydr380w delta ydl080c delta - CENPK609-11A MATa MAL2-8c SUC2 pdc1, 5, 6 delta ydr380w delta