Protein Sculptures: Bridging the Gap Between Art and Science

Indianapolis, IN

THI3 is a putative regulatory protein in Saccharomyces cerevisiae which, while possessing a great deal of sequence identity with the thiamin diphosphate (ThDP)-dependent pyruvate decarboxylase (PDC) from the same species, appears to have no PDC activity. Instead, it seems to have evolved a function in the genetic regulation of thiamin biosynthesis that is linked to its binding intracellular ThDP. Interestingly, the active sites of THI3 and PDC are essentially conserved with the exceptions of the substitution of a glutamate in PDC for an aspartate in THI3, of an aspartate in PDC for a glutamate in THI3, and of an isoleucine in PDC for a valine in THI3. In PDC, the active site glutamate and aspartate have been shown to act as critical catalytic acid-base groups, while the isoleucine residue is involved in holding the ThDP cofactor in the strained conformation necessary for the formation of the reactive ylide. Due to their similar properties, it is surprising that the amino acid substitutions in THI3 would result in the complete loss of PDC activity and the evolution of a unique regulatory functionality. In order to better understand the mechanistic factors responsible for these results, site-directed mutagenesis has been attempted to engineer PDC activity into THI3 through the generation of the various single, double, and triple mutants. These mutants are currently in the process of being expressed, purified, and characterized.