Enzymes are macromolecular machines in the cells that catalyze the chemistry of life. Structural and biochemical studies of enzymes and its ligands shed lights on the catalytic mechanism and guide efforts of rational drug design. In this work, threedimensional structures and biochemical analysis are presented to expand our understanding of two independent enzymes, pseudouridine monophosphate ([PSI]MP) glycosidase and thiamine pyrimidine synthase. Pseudouridine ([PSI]), the C-isomer of uridine, is the most abundant modified nucleotide in nature. Despite extensive mechanistic study of [PSI] biosynthesis, the degradation pathway for [PSI] has not been discovered until recently. In this pathway, [PSI] is first phosphorylated by [PSI] kinase and then degraded into uracil and ribose 5phosphate by [PSI]MP glycosidase. Here we present crystallographic and biochemical study of [PSI]MP glycosidase. Four unique structures of [PSI]MP glycosidase were displayed to illustrate the reaction pathway of [PSI]MP degradation. The combination of structural and biochemical study elucidates the mechanism of [PSI] degradation and stands as the first example of the mechanistic study of the unusual C-C glycosidic bond cleavage. Thiamin pyrophosphate is the active form of vitamin B1 and an essential cofactor for all living systems. The synthesis of thiamin pyrophosphate involves coupling of the thiazole and pyrimidine moieties, which are synthesized separately. All enzymes in prokaryotes and eukaryotes involved in the thiamin biosynthesis pathway have been structurally characterized, except the eukaryotic thiamin pyrimidine synthase THI5p. THI5p synthesizes the pyrimidine moiety of thiamin from histidine and pyridoxal phosphate. This study suggested that THI5p served as the histidine source and became inactive after a single turnover. Moreover, crystal structure of THI5p revealed the identity of the histidine residue and the spatial relationship between the imidazole and the pyridoxal ring. As the reaction also required iron and oxygen, a starting mechanism was proposed, involving Diels-Alder reaction followed by iron-mediated oxidation. Additional structural evidence showed oxidative modification of a cysteine residue at the potential iron binding site, in THI5p after the reaction. The irreversible modification of the cysteine thiol provided further insights into the remarkable chemistry of this suicide enzyme.