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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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Enzymatic characterization of St AckA. Plot of enzyme activity (%) at various (A) pH and (B) Temperature values. (C) Profile of saturation kinetics exhibited by StAckA with acetate as substrate.
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Figure 2: Enzymatic characterization of St AckA. Plot of enzyme activity (%) at various (A) pH and (B) Temperature values. (C) Profile of saturation kinetics exhibited by StAckA with acetate as substrate.

Mentions: Earlier studies on SCFA specificity of AckAs have led to conflicting reports [5,9]. In the present study, the recombinant StAckA was purified to homogeneity using Ni-NTA affinity chromatography followed by gel-filtration. The preliminary enzymatic assay with acetate, ATP and Mg2+ as substrates showed that the StAckA catalyzed reaction follows Michaelis-Menten kinetics with pH and temperature optima in the range of 7.2-7.5 and 35–37°C, respectively (Figure 2). This highly purified, stable and active preparation of StAckA was further used to examine the kinetics of phosphoryl transfer reaction from ATP to various SCFA substrates viz. formate, acetate, propionate and butyrate. It was observed that StAckA could catalyze phosphate transfer at significant rate from ATP to formate, acetate and propionate (Table 1). However, butyrate was not found to be a suitable substrate. In addition, the presence of butyrate at equimolar concentration reduced the catalytic rate of acetate utilization by 40%, which might suggest competitive binding of butyrate at the SCFA binding site. Further, kinetic analysis showed that the Km is nearly 10 times lower for acetate when compared to formate or propionate (Table 1). As maximum velocity and catalytic turnover numbers (kcat) were found to be similar for these substrates, the catalytic efficiency (kcat/Km) for acetate was ~10 times higher than for formate or propionate, indicating that acetate is the preferred substrate. Noticeably, the enzyme showed similar Km and kcat values for ATP when either formate, acetate or propionate was used as the SCFA substrate (Table 1). Further kinetic characterization revealed that StAckA catalyzes the reverse reaction i.e. phosphoryl transfer from acetyl-phosphate to ADP much more efficiently (Table 1). The Km for ADP and ATP are comparable whereas the Km for acetyl-phosphate is significantly lower compared to that for acetate.


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)

Enzymatic characterization of St AckA. Plot of enzyme activity (%) at various (A) pH and (B) Temperature values. (C) Profile of saturation kinetics exhibited by StAckA with acetate as substrate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Enzymatic characterization of St AckA. Plot of enzyme activity (%) at various (A) pH and (B) Temperature values. (C) Profile of saturation kinetics exhibited by StAckA with acetate as substrate.
Mentions: Earlier studies on SCFA specificity of AckAs have led to conflicting reports [5,9]. In the present study, the recombinant StAckA was purified to homogeneity using Ni-NTA affinity chromatography followed by gel-filtration. The preliminary enzymatic assay with acetate, ATP and Mg2+ as substrates showed that the StAckA catalyzed reaction follows Michaelis-Menten kinetics with pH and temperature optima in the range of 7.2-7.5 and 35–37°C, respectively (Figure 2). This highly purified, stable and active preparation of StAckA was further used to examine the kinetics of phosphoryl transfer reaction from ATP to various SCFA substrates viz. formate, acetate, propionate and butyrate. It was observed that StAckA could catalyze phosphate transfer at significant rate from ATP to formate, acetate and propionate (Table 1). However, butyrate was not found to be a suitable substrate. In addition, the presence of butyrate at equimolar concentration reduced the catalytic rate of acetate utilization by 40%, which might suggest competitive binding of butyrate at the SCFA binding site. Further, kinetic analysis showed that the Km is nearly 10 times lower for acetate when compared to formate or propionate (Table 1). As maximum velocity and catalytic turnover numbers (kcat) were found to be similar for these substrates, the catalytic efficiency (kcat/Km) for acetate was ~10 times higher than for formate or propionate, indicating that acetate is the preferred substrate. Noticeably, the enzyme showed similar Km and kcat values for ATP when either formate, acetate or propionate was used as the SCFA substrate (Table 1). Further kinetic characterization revealed that StAckA catalyzes the reverse reaction i.e. phosphoryl transfer from acetyl-phosphate to ADP much more efficiently (Table 1). The Km for ADP and ATP are comparable whereas the Km for acetyl-phosphate is significantly lower compared to that for acetate.

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