Browsing by Subject "Thiamin Diphosphate-Dependent Enzymes"

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    Investigation of the Evolutionary Aspects of Thiamin Diphosphate-Dependent Decarboxylases
    (2015) Rogers, Megan P.; McLeish, Michael J.
    Thiamin diphosphate (ThDP)-dependent enzymes catalyze a wide range of reactions including the oxidative and nonoxidative decarboxylation of 2-keto acids, carboligation reactions, the cleavage of C-C bonds, and the formation of C-S, C-N, and C-O bonds. Surprisingly, given this diversity, all ThDP-dependent enzyme catalyzed reactions proceed through essentially the same intermediate. This suggests that these enzymes share a common ancestry and have evolved to become the diverse group of enzymes seen today. Sequence alignments have revealed that all ThDP-dependent enzymes share two common ThDP binding domains, the PYR domain and the PP domain. In addition to these conserved domains, over time, other domains have been added creating further diversity in this superfamily. For instance, the TH3 domain, found in many ThDP-dependent enzymes, serves the function of binding additional cofactors such as FAD in enzymes like acetohydroxyacid synthase (AHAS) but in others, like pyruvate decarboxylase (PDC), it has lost this function completely. The work presented here focuses on ThDP-dependent decarboxylases. In this thesis, several evolutionary aspects of this group of enzymes will be examined including (i) the characterization of an evolutionary forerunner in the presence of a mechanism-based inhibitor, (ii) the characterization of the minor isozymes of pyruvate decarboxylase from Saccharomyces cerevisiae, and (iii) the development of a selection method to increase the efficiency of the site-saturation mutagenesis used to study ThDP-dependent enzyme evolution.