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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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Conformational changes induced upon ligand-binding in AckAs. (A) Structural comparison of A-subunit of Form-I StAckA (apo, pink) with open (yellow) and closed (cyan) subunits of MtAckA (PDB:1TUY). A large movement in domain-I (moving domain) relative to the domain-II (fixed domain) could be observed. Regions connecting the two domains are represented by the putative hinge residues (Ala152 and Thr385). N- and C-termini as well as secondary structures corresponding to the core helices are labeled. (B) Influence of ligand binding on the intrinsic fluorescence (excitation: 280 nm, emission: 300–400 nm) of StAckA.
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Figure 5: Conformational changes induced upon ligand-binding in AckAs. (A) Structural comparison of A-subunit of Form-I StAckA (apo, pink) with open (yellow) and closed (cyan) subunits of MtAckA (PDB:1TUY). A large movement in domain-I (moving domain) relative to the domain-II (fixed domain) could be observed. Regions connecting the two domains are represented by the putative hinge residues (Ala152 and Thr385). N- and C-termini as well as secondary structures corresponding to the core helices are labeled. (B) Influence of ligand binding on the intrinsic fluorescence (excitation: 280 nm, emission: 300–400 nm) of StAckA.

Mentions: Proteins belonging to ASKHA superfamily have been suggested to undergo inter-domain motion upon ligand binding [30-33] (Figure 5A). In the present study, ligand binding to StAckA was examined by monitoring the thermal stability of the enzyme using CD spectroscopy. The unliganded enzyme showed a melting temperature (Tm) of 45°C at which 50% of the enzyme was unfolded. An increased Tm was observed with the addition of acetate (50°C), acetyl-phosphate (54°C), ADP (52°C) and ATP (52°C), suggesting that the protein becomes more stable upon ligand binding. Noticeably, StAckA contains a single tryptophan (Trp46) residue, which is located in domain-I at a large distance (20 Å between Cα of Trp46 and Asn213, a residue involved in binding of nucleotide phosphates) from the active site. This allows monitoring of conformational transition induced upon ligand-binding by measuring fluorescence quenching due to the change in the tryptophan environment. Intrinsic fluorescence spectra of StAckA recorded in the presence of acetate, acetyl-phosphate, ADP and ATP showed quenching as compared to the unliganded enzyme (Figure 5B), thus indicating that binding of these ligands is likely to result in conformational transitions that may involve inter-domain movement.


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)

Conformational changes induced upon ligand-binding in AckAs. (A) Structural comparison of A-subunit of Form-I StAckA (apo, pink) with open (yellow) and closed (cyan) subunits of MtAckA (PDB:1TUY). A large movement in domain-I (moving domain) relative to the domain-II (fixed domain) could be observed. Regions connecting the two domains are represented by the putative hinge residues (Ala152 and Thr385). N- and C-termini as well as secondary structures corresponding to the core helices are labeled. (B) Influence of ligand binding on the intrinsic fluorescence (excitation: 280 nm, emission: 300–400 nm) of StAckA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 5: Conformational changes induced upon ligand-binding in AckAs. (A) Structural comparison of A-subunit of Form-I StAckA (apo, pink) with open (yellow) and closed (cyan) subunits of MtAckA (PDB:1TUY). A large movement in domain-I (moving domain) relative to the domain-II (fixed domain) could be observed. Regions connecting the two domains are represented by the putative hinge residues (Ala152 and Thr385). N- and C-termini as well as secondary structures corresponding to the core helices are labeled. (B) Influence of ligand binding on the intrinsic fluorescence (excitation: 280 nm, emission: 300–400 nm) of StAckA.
Mentions: Proteins belonging to ASKHA superfamily have been suggested to undergo inter-domain motion upon ligand binding [30-33] (Figure 5A). In the present study, ligand binding to StAckA was examined by monitoring the thermal stability of the enzyme using CD spectroscopy. The unliganded enzyme showed a melting temperature (Tm) of 45°C at which 50% of the enzyme was unfolded. An increased Tm was observed with the addition of acetate (50°C), acetyl-phosphate (54°C), ADP (52°C) and ATP (52°C), suggesting that the protein becomes more stable upon ligand binding. Noticeably, StAckA contains a single tryptophan (Trp46) residue, which is located in domain-I at a large distance (20 Å between Cα of Trp46 and Asn213, a residue involved in binding of nucleotide phosphates) from the active site. This allows monitoring of conformational transition induced upon ligand-binding by measuring fluorescence quenching due to the change in the tryptophan environment. Intrinsic fluorescence spectra of StAckA recorded in the presence of acetate, acetyl-phosphate, ADP and ATP showed quenching as compared to the unliganded enzyme (Figure 5B), thus indicating that binding of these ligands is likely to result in conformational transitions that may involve inter-domain movement.

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