Reversible lysine acetylation in Streptomyces lividans and Salmonella enterica
Tucker, Alex Clayton
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Lysine acetylation is a post-translational modification conserved in all domains of life. Lysine acetylation occurs lysine side chains and affects functions of proteins involved in diverse cellular pathways including transcription, chromatin maintenance, cell structure, and central metabolism. Acetyltransferase enzymes carry out acetylation of specific lysine residues in target proteins. The protein acetyltransferase Pat enzymes from the γ-proteobacterium Salmonella enterica and the α-proteobacterium Rhodopseudomonas palustris contain 2 domains: an N-terminal domain of unknown function and a C-terminal GNAT domain. S. enterica Pat and R. palustris Pat are the best-characterized bacterial protein acetyltransferases. Pat homologs, along with protein deacetylases, regulate the activity of AMP-forming CoA ligases (ACLs) and structurally unrelated targets in vivo. The first part of this work describes the identification and characterization of two active isoforms of CobB sirtuin-type (NAD+-dependent) deacetylase expressed as part of the S. enterica reversible lysine acetylation system. Reversible lysine acetylation is poorly understood in Gram-positive bacteria. The actinomycete S. lividans encodes a Pat homolog, SlPatA, with reversed domain order (i.e. N¬-terminal GNAT domain and C-terminal domain of unknown function). Experiments here demonstrate that SlPatA regulates acetoacetyl-CoA synthetase (AacS), a member of the ACL family, in S. lividans. Acetylation of the active site lysine inactivates AacS. A proteomic-based study of acetylated proteins in S. lividans reveals that SlPatA regulates the acetylation of four additional ACL enzymes in S. lividans. These data suggest that a major role of SlPatA is to regulate ACL enzymes. These studies also describe how SlPatA recognizes a protein substrate. SlPatA does not efficiently acetylate the S. lividans ACL enzyme acetyl-CoA synthetase (Acs) in vitro. By swapping regions of S. lividans Acs protein with a known protein substrate of SlPatA, we generated a S. lividans Acs variant that is efficiently acetylated in vitro. This work defined elements in protein substrates recognized by SlPatA. Structure guided studies of the SlPatA GNAT domain, reveal important electro static interactions between the SlPatA GNAT domain and a protein substrate. A crosslinked complex composed of the GNAT and protein substrate was generated that likely resembles a biologically acetylation complex.