Genetic analysis of thiamine metabolism in Saccharomyces cerevisiae

A genetic analysis of thiamine metabolism has been carried out in the budding yeast, Saccharomyces cerevisiae. A collection of thiamine auxotrophic mutants were isolated following UV and Ty insertion mutagenesis. The mutations responsible for the auxotrophic phenotypes were characterised to different extents through complementation analysis, molecular cloning and enzyme assays. In total 171 mutants were analysed and all of these have been assigned to complementation groups, genes and/or functions. Some newly isolated mutations were found to be allelic with the known biosynthetic genes, THI4 and THI6 others were in the regulatory genes, THI2 and THI3 two more defined a new function for the transcription factor, Pdc2p, namely thiamine gene activation. In addition the previously known mutations, thil, thi2, and thi3, were complemented and the sequences of the wild-type THI1, THI2 and THI3 genes were found. From the deduced amino acid sequences roles for the gene products were hypothesised. The Thi2p was found to be homologous with the Gal4p transcription factor due to the presence of a Zn-finger motif therefore a DNA-binding transcription factor role was proposed for this protein. The Thi3p was found to be homologous to the structural proteins for the enzyme pyruvate decarboxylase. It contains a conserved sequence for TPP binding, the consensus motif having been found in all TPP-dependent enzymes. Therefore it is hypothesised here that Thi3p acts as a "TPP sensor" within the cell, such that deactivation of thiamine-regulated genes is exerted when TPP is bound to Thi3p. In the case of THI1, the complementing ORF was found to be a previously characterised gene, ILV2, which encodes the aceto-hydroxy acid synthase (AHAS) enzyme. AHAS catalyses the first step in the parallel biosyntheses of the branched-chain amino acids, isoleucine and valine, using TPP as a cofactor. It was hypothesised that thil encodes a functional AHAS which has a reduced affinity for TPP resulting in a thiamine auxotrophic phenotype. The thil allele was cloned and enzyme assays were carried out which supported this hypothesis. Sequencing analysis and site-directed mutagenesis revealed that the thil phenotype was produced as a result of a single point mutation which caused the conserved amino acid substitution D176E. Hence in this study an amino acid residue potentially important in the binding of TPP to the AHAS enzyme is identified.