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

Chittori S, Savithri HS, Murthy MR - BMC Struct. Biol. (2012)

Bottom Line: These domains adopt an intermediate conformation compared to that of open and closed forms of ligand-bound Methanosarcina thermophila AckA (MtAckA).Unexpectedly, Form-II StAckA structure showed a drastic change in the conformation of residues 230-300 compared to that of Form-I.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka 560012, India.

ABSTRACT

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 (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 k(cat)/K(m) 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 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 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.

Conclusions: The 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.

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Interactions of the citrate bound at the dimeric interface of Form-II St AckA. Stereo diagram of the putative ligand-binding pocket at the dimeric interface showing bound citrate (CIT-501, magenta). Citrate anchoring residues contributed by subunit-A (pale-yellow) and subunit-B (cyan) are shown in ball and stick representation. Electron density contoured at 1σ (2Fo-Fc) is also shown
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Figure 7: Interactions of the citrate bound at the dimeric interface of Form-II St AckA. Stereo diagram of the putative ligand-binding pocket at the dimeric interface showing bound citrate (CIT-501, magenta). Citrate anchoring residues contributed by subunit-A (pale-yellow) and subunit-B (cyan) are shown in ball and stick representation. Electron density contoured at 1σ (2Fo-Fc) is also shown

Mentions: Examination of the difference electron density map of Form-II StAckA showed significant uninterpreted density located at the dimeric interface that was distinct from that of the polypeptide chain. Citrate, a component of the crystallization condition, was found to best fit the density (Figure 7) and could be refined with reasonable B-factors (33.9 Å2) and occupancy (0.75). Interestingly, only one citrate binds at the dimeric interface resulting in an asymmetric dimer. Citrate occupies a highly polar and charged environment. It interacts with atoms (distance cutoff: ≤5.0 Å) of residues Arg178, Asp227, Thr228, Ser229, Met230, Asp257, Thr258, Leu259, Gly260, Arg309, Lys312 and Tyr313 of the A-subunit and Arg309, Lys312 and Tyr313 of the B-subunit. Some of the residues that interact with the citrate are from the amino-terminal end of the variable segment and hence the conformational differences between the two subunits observed in the Form-II structure could be due to citrate binding.


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

Chittori S, Savithri HS, Murthy MR - BMC Struct. Biol. (2012)

Interactions of the citrate bound at the dimeric interface of Form-II St AckA. Stereo diagram of the putative ligand-binding pocket at the dimeric interface showing bound citrate (CIT-501, magenta). Citrate anchoring residues contributed by subunit-A (pale-yellow) and subunit-B (cyan) are shown in ball and stick representation. Electron density contoured at 1σ (2Fo-Fc) is also shown
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475010&req=5

Figure 7: Interactions of the citrate bound at the dimeric interface of Form-II St AckA. Stereo diagram of the putative ligand-binding pocket at the dimeric interface showing bound citrate (CIT-501, magenta). Citrate anchoring residues contributed by subunit-A (pale-yellow) and subunit-B (cyan) are shown in ball and stick representation. Electron density contoured at 1σ (2Fo-Fc) is also shown
Mentions: Examination of the difference electron density map of Form-II StAckA showed significant uninterpreted density located at the dimeric interface that was distinct from that of the polypeptide chain. Citrate, a component of the crystallization condition, was found to best fit the density (Figure 7) and could be refined with reasonable B-factors (33.9 Å2) and occupancy (0.75). Interestingly, only one citrate binds at the dimeric interface resulting in an asymmetric dimer. Citrate occupies a highly polar and charged environment. It interacts with atoms (distance cutoff: ≤5.0 Å) of residues Arg178, Asp227, Thr228, Ser229, Met230, Asp257, Thr258, Leu259, Gly260, Arg309, Lys312 and Tyr313 of the A-subunit and Arg309, Lys312 and Tyr313 of the B-subunit. Some of the residues that interact with the citrate are from the amino-terminal end of the variable segment and hence the conformational differences between the two subunits observed in the Form-II structure could be due to citrate binding.

Bottom Line: These domains adopt an intermediate conformation compared to that of open and closed forms of ligand-bound Methanosarcina thermophila AckA (MtAckA).Unexpectedly, Form-II StAckA structure showed a drastic change in the conformation of residues 230-300 compared to that of Form-I.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka 560012, India.

ABSTRACT

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 (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 k(cat)/K(m) 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 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 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.

Conclusions: The 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.

Show MeSH
Related in: MedlinePlus