Abstract
BackgroundBacteria such as Escherichia coli and Salmonella typhimurium can utilize acetate as the sole source of carbon and energy. Acetate kinase (AckA) and phosphotransacetylase (Pta), key enzymes of acetate utilization pathway, regulate flux of metabolites in glycolysis, gluconeogenesis, TCA cycle, glyoxylate bypass and fatty acid metabolism.ResultsHere we report kinetic characterization of S. typhimurium AckA (StAckA) and structures of its unliganded (Form-I, 2.70 Å resolution) and citrate-bound (Form-II, 1.90 Å resolution) forms. The enzyme showed broad substrate specificity with kcat/Km in the order of acetate > propionate > formate. Further, the Km for acetyl-phosphate was significantly lower than for acetate and the enzyme could catalyze the reverse reaction (i.e. ATP synthesis) more efficiently. ATP and Mg2+ could be substituted by other nucleoside 5′-triphosphates (GTP, UTP and CTP) and divalent cations (Mn2+ and Co2+), respectively. Form-I StAckA represents the first structural report of an unliganded AckA. StAckA protomer consists of two domains with characteristic βββαβαβα topology of ASKHA superfamily of proteins. These domains adopt an intermediate conformation compared to that of open and closed forms of ligand-bound Methanosarcina thermophila AckA (MtAckA). Spectroscopic and structural analyses of StAckA further suggested occurrence of inter-domain motion upon ligand-binding. Unexpectedly, Form-II StAckA structure showed a drastic change in the conformation of residues 230–300 compared to that of Form-I. Further investigation revealed electron density corresponding to a citrate molecule in a pocket located at the dimeric interface of Form-II StAckA. Interestingly, a similar dimeric interface pocket lined with largely conserved residues could be identified in Form-I StAckA as well as in other enzymes homologous to AckA suggesting that ligand binding at this pocket may influence the function of these enzymes.ConclusionsThe biochemical and structural characterization of StAckA reported here provides insights into the biochemical specificity, overall fold, thermal stability, molecular basis of ligand binding and inter-domain motion in AckA family of enzymes. Dramatic conformational differences observed between unliganded and citrate-bound forms of StAckA led to identification of a putative ligand-binding pocket at the dimeric interface of StAckA with implications for enzymatic function.
Highlights
Bacteria such as Escherichia coli and Salmonella typhimurium can utilize acetate as the sole source of carbon and energy
S. typhimurium AckA (StAckA) shares sequence identities of 41% and 40% with StTdcD and StPduW, respectively
An enzyme corresponding to PduW of S. typhimurium could not be identified in the closely related E. coli
Summary
Bacteria such as Escherichia coli and Salmonella typhimurium can utilize acetate as the sole source of carbon and energy. Acetate kinase (AckA) and phosphotransacetylase (Pta), key enzymes of acetate utilization pathway, regulate flux of metabolites in glycolysis, gluconeogenesis, TCA cycle, glyoxylate bypass and fatty acid metabolism. Utilization of acetate as a source of carbon and energy requires its initial activation to acetyl-CoA [1,2,3]. Sequential activities of acetate kinase (AckA, EC 2.7.2.1) and phosphotransacetylase (Pta, EC 2.3.1.8) result in the interconversion of ATP, acetate and CoA to ADP, acetyl-CoA and orthophosphate via acetylphosphate [4,5]. In addition to being an important pathway for carbon flow, the AckA-Pta activity might control the cellular concentration of acetyl-CoA and acetylphosphate, which serve as important metabolic intermediates. Studies on AckA from several organisms have shown that the enzyme is important for xylose metabolism, phosphoryl transfer to enzyme-I of the phosphoenolpyruvate: glucose phosphotransferase system, periplasmic binding proteins and response regulator proteins of two-component systems [8,9,10,11]
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