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Background  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.    Results  Here we report kinetic characterization of  S. typhimurium  AckA ( St AckA) 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  k   cat  / K   m   in the order of acetate > propionate > formate. Further, the  K   m   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 Mg 2+  could be substituted by other nucleoside 5′-triphosphates (GTP, UTP and CTP) and divalent cations (Mn 2+  and Co 2+ ), respectively. Form-I  St AckA represents the first structural report of an unliganded AckA.  St AckA 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 ( Mt AckA). Spectroscopic and structural analyses of StAckA further suggested occurrence of inter-domain motion upon ligand-binding. Unexpectedly, Form-II  St AckA 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  St AckA. Interestingly, a similar dimeric interface pocket lined with largely conserved residues could be identified in Form-I  St AckA as well as in other enzymes homologous to AckA suggesting that ligand binding at this pocket may influence the function of these enzymes .Conclusions  The biochemical and structural characterization of  St AckA 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  St AckA led to identification of a putative ligand-binding pocket at the dimeric interface of  St AckA with implications for enzymatic function.

bmc structural biology

Structural and mechanistic investigations on Salmonella typhimurium acetate kinase (AckA): identification of a putative ligand binding pocket at the dimeric interface