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Abstract
Acetylation is a chemical modification conserved in all domains of life. Acetylation has been dominantly defined as a post-translational modification of lysyl residues of proteins carried out by Gcn5 N-acetyltransferases (GNATs, PF 00583). Within a single organism, a genome can code for 7-70 acetyltransferases, all with sequence similarity that currently makes it impossible to determine substrate specificity. There has recently been an upsurge of acetyltransferases that target amine groups of diverse small molecule substrates, rather than proteins, generating an additional layer of complexity in characterizing putative acetyltransferases. In most cases, acetylation of proteins alters the enzymes activity, providing the cell with a rapid mechanism to adapt to external or internal fluctuations. The protein acetylation paradigm in prokaryotes involves the modification of an active site lysyl residue of acetyl-CoA synthetase (Acs). In many prokaryotic species, Acs is acetylated by a single protein acetyltransferase containing a regulatory domain, that depending on the organism, responds to amino acids, cAMP, NADP+, or unknown molecules. The cell has adapted to require Acs acetylation, for without it, cellular homeostasis is disturbed through improper control of ATP synthesis. Due to the fact that all organisms carry out acetylation, this thesis focuses on determining acetyltransferase substrate preferences in a variety of prokaryotic organisms. Through this work, acetyltransferases have been characterized in Salmonella enterica, Streptomyces lividans, and Rhodopseudomonas palustris. In S. enterica, characterization of a toxin protein acetyltransferase led to insight into how the cell enters persistence during infection. Additionally, using a S. enterica model for phosphinothricin detoxification, acetyltransferases from Deinococcous radiodurans, Geobacillus kaustophilus, Bacillus subtilis, Burkholderia xenovorans, Staphylococcus aureus, Acinetobacter baylyi, and Escherichia coli were characterized as small molecule acetyltransferases that detoxified a myriad of methionine derivatives. In S. lividans, Acs acetylation was found to be unique in that it required serine acetylation within the active site. Lastly, in R. palustris, small molecule acetylation of aminobenzoates was essential to benzoate catabolism and synthesis of reaction center proteins. Although the projects are diverse, each answers unique questions about protein and small molecule acetylation in prokaryotic physiology.