| Summary: | Regulatory protein phosphorylation is a well conserved mechanism of signal transduction in all biological systems. In bacteria, signal transduction by phosphorylation is thought to occur primarily on His and Asp residues. However, phosphoproteomic surveys in phylogenetically diverse bacteria over the past decade have identified numerous proteins that are phosphorylated on serine (Ser) and/or threonine (Thr) residues. Consistently, genes encoding Ser/Thr kinases are present in many bacterial genomes such as E. coli, which encodes at least three Ser/Thr kinases. Since Ser/Thr phosphorylation is a stable modification, a dedicated phosphatase is necessary to allow reversible regulation. Bacterial Ser/Thr phosphatases which have extensive sequence and structural homology to eukaryotic Ser/Thr PP2C-type phosphatases are referred to as eukaryotic-like Ser/Thr phosphatases (eSTPs). eSTPs have been identified in a number of bacteria, but none have been reported in E. coli. The work presented in this thesis was aimed at identifying and biochemically characterizing a eukaryotic-like Ser/Thr phosphatase and its partner Ser/Thr kinase in E.coli.
Chapter 3 describes the identification of a novel PP2C-like Ser/Thr phosphatase PphC encoded by an E. coli ORF, yegK, and characterization of its biochemical properties including kinetics, substrate specificity and sensitivity to known phosphatase inhibitors. I investigated differences in the activity of this protein in closely related E. coli strains. Finally, I demonstrated that this eSTP acts to dephosphorylate a novel Ser/Thr kinase which is encoded in the same operon suggesting that they most likely function as a pair in regulating Ser/Thr phosphorylation.
Chapter 4 describes the biochemical characterization of a Ser/Thr kinase YegI in E. coli. I show that YegI is an active kinase with significant structural homology to eukaryotic Ser/Thr kinases. The YegI kinase domain is tethered to a cytoplasmic C-terminal domain containing two non-specific DNA binding Helix-hairpin helix motifs. I have identified enolase and elongation factor-Tu (EF-Tu) as potential physiological substrates of YegI and have demonstrated that phosphorylation of EF-Tu by YegI inhibits protein translation in vitro.
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