Limits...
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.

Show MeSH

Related in: MedlinePlus

Molecular basis for the substrate and nucleotide specificity of St AckA. (A) Stereo diagram of SCFA binding residues (lime-green) of StAckA with modeled acetate (grey). For comparison, a few superposed residues (teal) of MtAckA are also shown. (B) Stereo diagram of the putative nucleotide-binding residues (salmon-red) of StAckA and modeled ADP (grey). Distances shown with dashed lines are in Å.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3475010&req=5

Figure 4: Molecular basis for the substrate and nucleotide specificity of St AckA. (A) Stereo diagram of SCFA binding residues (lime-green) of StAckA with modeled acetate (grey). For comparison, a few superposed residues (teal) of MtAckA are also shown. (B) Stereo diagram of the putative nucleotide-binding residues (salmon-red) of StAckA and modeled ADP (grey). Distances shown with dashed lines are in Å.

Mentions: As observed from kinetic measurements, StAckA could catalyze phosphoryl transfer to formate, acetate and propionate but not to butyrate (Table 1). Despite several attempts, crystals of ligand bound enzyme could not be obtained either by co-crystallization or soaking. Therefore, based on the acetate-AlF3-ADP bound structure of MtAckA [16], formate, acetate, propionate and butyrate were modeled into the active site of the Salmonella enzyme. As anticipated, acetate could be accommodated with favorable interactions (Figure 4A). Although formate could also be accommodated, it made fewer interactions when compared to that of acetate while the additional methyl group of propionate (as compared to acetate) formed a short contact with Val93 of StAckA. This short contact could however be relieved by a small conformational adjustment of Val93 or by pointing the extra methyl-group of propionate towards Ala181, which is a semi-conserved residue and is mostly replaced by phenylalanine in acetokinases (Figures 1 and 4A). The aromatic ring of this phenylalanine bends away from the active site pocket and thus effectively presents a hydrophobic environment equivalent to that of alanine towards the SCFA binding site (Figure 4A). Similar adjustment with butyrate was not possible as the short contacts with Val93 and Ala181 could not be relieved by small conformational adjustments that would allow the reactive group of butyrate to be oriented appropriately for catalysis.


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)

Molecular basis for the substrate and nucleotide specificity of St AckA. (A) Stereo diagram of SCFA binding residues (lime-green) of StAckA with modeled acetate (grey). For comparison, a few superposed residues (teal) of MtAckA are also shown. (B) Stereo diagram of the putative nucleotide-binding residues (salmon-red) of StAckA and modeled ADP (grey). Distances shown with dashed lines are in Å.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Molecular basis for the substrate and nucleotide specificity of St AckA. (A) Stereo diagram of SCFA binding residues (lime-green) of StAckA with modeled acetate (grey). For comparison, a few superposed residues (teal) of MtAckA are also shown. (B) Stereo diagram of the putative nucleotide-binding residues (salmon-red) of StAckA and modeled ADP (grey). Distances shown with dashed lines are in Å.
Mentions: As observed from kinetic measurements, StAckA could catalyze phosphoryl transfer to formate, acetate and propionate but not to butyrate (Table 1). Despite several attempts, crystals of ligand bound enzyme could not be obtained either by co-crystallization or soaking. Therefore, based on the acetate-AlF3-ADP bound structure of MtAckA [16], formate, acetate, propionate and butyrate were modeled into the active site of the Salmonella enzyme. As anticipated, acetate could be accommodated with favorable interactions (Figure 4A). Although formate could also be accommodated, it made fewer interactions when compared to that of acetate while the additional methyl group of propionate (as compared to acetate) formed a short contact with Val93 of StAckA. This short contact could however be relieved by a small conformational adjustment of Val93 or by pointing the extra methyl-group of propionate towards Ala181, which is a semi-conserved residue and is mostly replaced by phenylalanine in acetokinases (Figures 1 and 4A). The aromatic ring of this phenylalanine bends away from the active site pocket and thus effectively presents a hydrophobic environment equivalent to that of alanine towards the SCFA binding site (Figure 4A). Similar adjustment with butyrate was not possible as the short contacts with Val93 and Ala181 could not be relieved by small conformational adjustments that would allow the reactive group of butyrate to be oriented appropriately for catalysis.

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