Mechanistic Enzymology Of Plasmodium Falciparum Sir2, Structural And Biochemical Studies On Nicotinamidases And The Structure And Function Of Enzymes In The Purine Degradative Pathway From Klebsiella Pneumoniae

摘要

Enzymes are proteins that catalyze the chemistry of life. Biochemical and structural characterization of an enzyme and the interactions it makes with ligands facilitates our understanding of the processes that make life possible and enables us to devise methods to modulate the enzyme's activity for the benefit of human health. In this work, biochemical and structural studies are presented that aim to expand our understanding of several classes of enzymes. In the first part, analyses of two enzymes that have been shown to have a role in lifespan extension in some organisms are presented. The Sir2 enzyme from the malaria-causing parasite Plasmodium falciparum is shown to be an NAD+-dependent deacetylase and an acetyllysinedependent and acetyllysine-independent NAD+ glycohydrolase. In addition, several examples of the Sir2 modulating enzyme, nicotinamidase, are characterized both structurally and biochemically. These works provided additional insights into and valuable tools for the study of Sir2-dependent lifespan extension. In the second part, several studies of enzymes that are responsible for purine degradation in the opportunistic human pathogen, Klebsiella pneumoniae, are presented. Using a combination of structural and biochemical methods, we characterize the enzymes responsible for ureidoglycine aminotransfer, 2-oxo-4-hydroxy-4-carboxy-5- ureidoimidazoline (OHCU) decarboxylase and allantoin racemase. The examination of the aminotransferase from K. pneumoniae reveals a novel pathway from ureidoglycine to oxalurate and provides structural evidence for this enzyme's specificity for ureidoglycine. We characterize the first reported inhibitor of OHCU decarboxylase and demonstrate structurally and biochemically how this molecule causes a conformational change, disrupting the organization of the active site. The structures of allantoin racemase, the first for this class of enzyme, shed new light on ligand binding and the mechanism of the racemization reaction. Together, these studies elucidate a great deal of the unique complexity that is present in this important pathway.