Determinants within the C-Terminal Domain of Streptomyces lividans Acetyl-CoA Synthetase that Block Acetylation of Its Active Site Lysine In Vitro by the Protein Acetyltransferase (Pat) Enzyme

Escalante-Semerena JC (2014) Determinants within the C-Terminal Domain of Streptomyces lividans Acetyl-CoA Synthetase that Block Acetylation of Its Active Site Lysine In Vitro by the Protein Acetyltransferase (Pat) Enzyme. PLoS ONE 9(6): e99817. doi:10.1371/journal.pone.0099817 Determinants within the C -Terminal Domain of Streptomyces lividans Acetyl-CoA Synthetase that Block Acetylation of Its Active Site Lysine In Vitro by the Protein Acetyltransferase (Pat) Enzyme Alex C. Tucker 0 Jorge C. Escalante-Semerena 0 Fernando Rodrigues-Lima, University Paris Diderot-Paris 7, France 0 Department of Microbiology, University of Georgia , Athens, Georgia , United States of America Reversible lysine acetylation (RLA) is a widespread regulatory mechanism that modulates the function of proteins involved in diverse cellular processes. A strong case has been made for RLA control exerted by homologues of the Salmonella enterica protein acetyltransferase (SePat) enzyme on the broadly distributed AMP-forming CoA ligase (a.k.a. acyl-CoA synthetases) family of metabolic enzymes, with acetyl-CoA synthetase (Acs) being the paradigm in the field. Here we investigate why the Acs homologue in Streptomyces lividans (SlAcs) is poorly acetylated in vitro by the S. lividans protein acetyltransferase (SlPat) enzyme. Chimeras of S. enterica Acs (SeAcs) and S. lividans Acs (SlAcs) constructed during the course of this work were acetylated by SlPatA in vitro, retained most of their activity, and were under RLA control in a heterologous host. We identified SeAcs residues N- and C-terminal to the target lysine that when introduced into SlAcs, rendered the latter under RLA control. These results lend further support to the idea that Pat enzymes interact with extensive surfaces of their substrates. Finally, we suggest that acetylation of SlAcs depends on factors or conditions other than those present in our in vitro system. We also discuss possible explanations why SlAcs is not controlled by RLA as defined in other bacterial species. - Funding: Funding was provided by United States Public Health Service, National Institutes of Health grant R01 GM062203. The funders had no role in the design, data collection and analysis, decision to publish or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Reversible lysine acetylation (RLA) is a post-translational modification that occurs in all domains of life [1] and affects diverse cellular processes and functions. Acetyltransferases transfer the acetyl moiety from acetyl-CoA to the e-amino group of the target lysine. Lysine acetylation can affect enzyme activity [2], protein stability [3], protein-protein interactions, or DNA binding [4]. Yeast Gcn5 protein (yGcn5p)-related N-acetyltransferases (a.k.a., GNATs), classified by amino acid sequence and structure [5], are the only class of acetyltransferases found in all domains of life [6]. GNATs were first identified for their role in modification of histones [7]. Crystal structures and biochemical analyses of the yGcn5p, the founding member of the GNAT family, with representative peptides from histones has provided valuable information about the substrate specificity and substrate recognition by GNATs [8,9]. Members of the GNAT family also acetylate metabolic enzymes. For example, in Salmonella enterica, the enzyme acetylCoA synthetase (SeAcs) is acetylated by the protein acetyltransferase (SePat), a two-domain acetyltransferase that contains a large domain of unknown function and a C-terminal GNAT domain [10]. SeAcs is a member of the AMP-forming CoA ligase family of enzymes that converts carboxylic acids to their CoA thioesters via an acyl-AMP intermediate [11]. Acetylation of the active site lysine of AMP-forming CoA ligases prevents the adenylylation of the carboxylic acid. In addition to Pat from S. enterica, GNATs are known to acetylate members of the of AMP-forming CoA ligase family (including Acs) in Rhodopseudomonas palustris [12,13], Bacillus subtilis [14], and Mycobacterium smegmatis [15]. The Acs homologue from Streptomyces coelicolor is acetylated in vivo [16], but the GNAT responsible for acetylation of S. coelicolor Acs is unknown. Knowledge of the interactions of GNAT with their proteins substrates is limited. R. palustris encodes a single-domain GNAT (RpKatA) and a homologue of the SePat GNAT (RpPat). RpKatA and RpPat discriminate among members of the AMP-forming CoA ligase family produced by R. palustris [13]. In addition to the target lysine, RpPat recognizes a loop greater than 20 A from the target lysine, suggesting that Pat enzymes interact with a large surface of the acceptor substrate [17]. As a proof of principle, the introduction of this recognition loop into R. palustris methylmalonyl-CoA mutase (RpMatB), an AMP-forming CoA ligase that is not a substrate of RpPat, rendered RpMatB a target of acetylation by RpPat. Thus, synthetic chimeras of AMP-forming CoA ligases have yielded valuable information about how GNATs recognize protein substrates and have produced AMP-forming CoA ligases that are placed under the regulation of lysine acetylation. RpPat and SePat enzymes acetylate their cognate Acs proteins. Although the GNAT responsible for the acetylation of Acs in S. coeolicolor is unknown, the closely related actinomycete Streptomyces lividans encodes SlPatA, a two-domain homologue of SePat and RpPat enzymes. Significantly, SlPatA does not efficiently acetylate the S. lividans Acs (SlAcs) in vitro [18], making this the first Acs enzyme that is not efficiently acetylated by a Pat acetyltransferase. In contrast, SlPatA efficiently acetylates SeAcs. Here we probe the amino acid sequences in SeAcs that rendered it a better substrate for SlPatA than SlAcs is. By replacing amino acids from SeAcs into the C-terminus of SlAcs, we constructed SlAcs-SeAcs chimeras that were efficiently acetylated by SlPatA. One SlAcs-SeAcs chimera contained 41 amino acid differences from SlAcs. As a result of these changes, the SlAcs-SeAcs chimera was subject to regulation by SlPatA. We used a heterologous model system to demonstrate that the SlAcs-SeAcs chimera was subject to RLA regulation in vivo by SlPatA. In sum, we identified regions in SeAcs that were critical for recognition by SlPatA, and transferring of these residues into the poor substrate SlAcs resulted in a SlAcs variant that was efficiently regulated by SlPatA. Materials and Methods Bacterial Strains and Growth Conditions All strains and plasmids used in this study are listed in Tables 1 and 2, respectively. Escherichia coli and Salmonella enterica strains were grown at 37uC in lysogeny broth (LB, Difco) [19] or no-carbon essential (NCE) minimal medium [20] supplemented with sodium acetate (10 mM), MgSO4 (1 mM), and ampicillin (100 mg ml21). When necessary, antibiotics were used at the following concentrations: ampicillin, 100 mg ml21; tetracycline, 10 mg ml21; chlorampheni (...truncated)