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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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Putative ligand binding pocket at the dimeric interface of St AckA. (A) Representation of the A-subunit (yellow except for the variable segment shown in bright-orange) of Form-II StAckA with the bound citrate (CIT-501, pink, ball and stick model) indicating the location of the cavity observed at the dimeric interface. Secondary structures corresponding to the variable segments are labeled. (B) A similar surface representation of the B-subunit (cyan with variable segment highlighted in blue) of Form-II StAckA. (C) Dimeric interface pocket in Form-I StAckA. A- and B-subunits of Form-I StAckA are shown in green and red, respectively. Active site cavity of each subunit is shown. Dimeric interface pocket is highlighted using spheres
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Figure 8: Putative ligand binding pocket at the dimeric interface of St AckA. (A) Representation of the A-subunit (yellow except for the variable segment shown in bright-orange) of Form-II StAckA with the bound citrate (CIT-501, pink, ball and stick model) indicating the location of the cavity observed at the dimeric interface. Secondary structures corresponding to the variable segments are labeled. (B) A similar surface representation of the B-subunit (cyan with variable segment highlighted in blue) of Form-II StAckA. (C) Dimeric interface pocket in Form-I StAckA. A- and B-subunits of Form-I StAckA are shown in green and red, respectively. Active site cavity of each subunit is shown. Dimeric interface pocket is highlighted using spheres

Mentions: Inspection of Form-II StAckA protomer structure revealed a shallow cavity at the site of citrate binding (Figures 8A and B). In the dimeric structure, these cavities coalesce, forming a larger pocket suitable for ligand binding (Figure 8C). Intriguingly, examination of Form-I StAckA as well as other structurally known members of acetokinase family (Table 3) revealed a similar cavity at the dimeric interface (Figure 8C and Additional file 2: Figure S2). As many of the residues (Arg178, Asp227, Met230, Arg309 and Tyr313; StAckA numbering) lining the cavity are highly conserved across acetokinases (indicated by green triangle in Figure 1), the dimeric interface pocket observed for the first time in Form-II StAckA could be involved in binding of a putative ligand that might influence the activity of the enzyme.


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)

Putative ligand binding pocket at the dimeric interface of St AckA. (A) Representation of the A-subunit (yellow except for the variable segment shown in bright-orange) of Form-II StAckA with the bound citrate (CIT-501, pink, ball and stick model) indicating the location of the cavity observed at the dimeric interface. Secondary structures corresponding to the variable segments are labeled. (B) A similar surface representation of the B-subunit (cyan with variable segment highlighted in blue) of Form-II StAckA. (C) Dimeric interface pocket in Form-I StAckA. A- and B-subunits of Form-I StAckA are shown in green and red, respectively. Active site cavity of each subunit is shown. Dimeric interface pocket is highlighted using spheres
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Putative ligand binding pocket at the dimeric interface of St AckA. (A) Representation of the A-subunit (yellow except for the variable segment shown in bright-orange) of Form-II StAckA with the bound citrate (CIT-501, pink, ball and stick model) indicating the location of the cavity observed at the dimeric interface. Secondary structures corresponding to the variable segments are labeled. (B) A similar surface representation of the B-subunit (cyan with variable segment highlighted in blue) of Form-II StAckA. (C) Dimeric interface pocket in Form-I StAckA. A- and B-subunits of Form-I StAckA are shown in green and red, respectively. Active site cavity of each subunit is shown. Dimeric interface pocket is highlighted using spheres
Mentions: Inspection of Form-II StAckA protomer structure revealed a shallow cavity at the site of citrate binding (Figures 8A and B). In the dimeric structure, these cavities coalesce, forming a larger pocket suitable for ligand binding (Figure 8C). Intriguingly, examination of Form-I StAckA as well as other structurally known members of acetokinase family (Table 3) revealed a similar cavity at the dimeric interface (Figure 8C and Additional file 2: Figure S2). As many of the residues (Arg178, Asp227, Met230, Arg309 and Tyr313; StAckA numbering) lining the cavity are highly conserved across acetokinases (indicated by green triangle in Figure 1), the dimeric interface pocket observed for the first time in Form-II StAckA could be involved in binding of a putative ligand that might influence the activity of the enzyme.

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