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A Study Of Novel Activities Of Type Iii Sirtuins: Cobb And Pfsir2A
Sirtuins are a class of nicotinamide adenine dinucleotides (NAD)-dependent histone deacetylases (HDACs) found in all three domains of life. Acetylation-deacetylation of histones lysine residues is an accepted paradigm for gene regulation in eukaryotes. In recent years, proteonomic studies revealed that many cellular proteins are acetylated and are regulated by post translational modifications. Although initially, sirtuins gained notoriety when Sir2 yeast was discovered to increase life span under calorie restriction, current research suggests that sirtuins are involved in a myriad of other cellular functions. The discovery of Sirt5's, a type III mammalian sirtuin,novel desuccinylase/demalonylase activity opened up new possibilities of cellular protein regulation. Two conserved active site residues, Tyr and Arg, are believed to be responsible for Sirt5's desuccinylase/demalonylase activity. The physiological importance of lysine succinylation and malonylation is strengthened by proteonomic studies that have identified succinylated and malonylated proteins. Many of those proteins are involved in metabolism and gene regulation suggesting that Sirt5 is important. Our aim was to verify the importance of the two conserved residues in type III sirtuins and investigate the physiological significance of these new post translational modifications in a bacterial model. We investigated the activity of two sirtuins, E. coli CobB, bears the two active site residues, and Plasmodium falciparum Sir2A (PfSir2A), a type III sirtuin missing the conserved residues, and found that only CobB has desuccinylase activity while PfSir2A prefers to remove long fatty acyl chains. We followed up on the physiological relevance of lysine succinylation by screening for CobB substrates in E. coli and found that CobB can regulate RcsB's DNA binding capability by lysine desuccinylation. We also investigated CobB's influence on metabolism in different nutrient conditions and found that desuccinylation was not important in acetate, succinate and propionate supplemented nutrients.
CobB; PfSir2A; Sirtuin
Ealick, Steven Edward; Cerione, Richard A
Chemistry and Chemical Biology
Ph.D. of Chemistry and Chemical Biology
Doctor of Philosophy
dissertation or thesis