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Abstract
In the post-genomic era, clinicians and scientists are increasingly reliant on interpretation of variants in metabolic genes for determining pathogenicity. These interpretations depend on functional annotation of the roles genes provide in metabolism, annotation which is far from complete. I embarked on a journey of enzyme discovery to fill gaps in our knowledge of metabolism in the budding yeast, Saccharomyces cerevisiae. I carried out a genetic and metabolomic screen of 120 uncharacterized candidate enzyme encoding genes that comprised my MSc. This dissertation describes my work in ascribing function to two distinct enzymes, Das2 and Tda5. Throughout my study I have found that Das2 is a novel uridine/cytidine kinase that functions in concert with a second minor uridine kinase, Urk1. These two enzymes are interdependent and in turn depend on a third enzyme, the major uracil phosphoribosyl transferase, Fur1 for stability. These three enzymes form a complex that is essential to wild-type pyrimidine salvage. As I aimed to elucidate the function of Tda5, I discovered that this uncharacterized enzyme is essential to growth. Loss of function mutations in TDA5 are alleviated by de-repression of its sporulation specific paralog, Ydl114w, and when YDL114W is deleted, tda5Δ is rescued by hypomorphic mutations in the ergosterol biosynthetic pathway. Analysis of metabolite levels in hand with nutritional sensitivities and genetic interaction data support the essential role of Tda5 in lipid homeostasis. Together, these studies assign new functions to previously uncharacterized genes and reveal functional interdependence of enzymes that sheds light on metabolic interpretations of genetic data.
