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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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Sequence analysis of acetokinase family of enzymes.  Multiple sequence alignment of St AckA with known structures belonging to acetokinase family. Sequence code: StAckA, S. typhimurium AckA; TmAckA, Thermotoga maritima AckA; MtAckA, Methanosarcina thermophila AckA; MaAckA, Mycobacterium avium AckA; FtTdcD, Francisella tularensis putative acetate/propionate kinase; StTdcD, S. typhimurium propionate kinase; TmBuk2, Thermotoga maritima butyrate kinase 2. All sequences are numbered at the beginning of each block of aligned sequences. StAckA numbering is indicated by every 10 residues using a dot symbol on top of the alignment. Secondary structures of Form-I StAckA and TmBuk2 (PDB:1SAZ) aligned onto their respective sequences are also shown (refer Figure 3A for secondary structure labeling scheme). Colour code: strictly conserved residues are shown in yellow with black background; highly similar regions are shown in blue with grey background. Putative acetate and nucleotide binding residues are marked with orange rhombi and magenta circles, respectively. Residues interacting with citrate in Form-II StAckA structure are highlighted by green triangles.
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Figure 1: Sequence analysis of acetokinase family of enzymes. Multiple sequence alignment of St AckA with known structures belonging to acetokinase family. Sequence code: StAckA, S. typhimurium AckA; TmAckA, Thermotoga maritima AckA; MtAckA, Methanosarcina thermophila AckA; MaAckA, Mycobacterium avium AckA; FtTdcD, Francisella tularensis putative acetate/propionate kinase; StTdcD, S. typhimurium propionate kinase; TmBuk2, Thermotoga maritima butyrate kinase 2. All sequences are numbered at the beginning of each block of aligned sequences. StAckA numbering is indicated by every 10 residues using a dot symbol on top of the alignment. Secondary structures of Form-I StAckA and TmBuk2 (PDB:1SAZ) aligned onto their respective sequences are also shown (refer Figure 3A for secondary structure labeling scheme). Colour code: strictly conserved residues are shown in yellow with black background; highly similar regions are shown in blue with grey background. Putative acetate and nucleotide binding residues are marked with orange rhombi and magenta circles, respectively. Residues interacting with citrate in Form-II StAckA structure are highlighted by green triangles.

Mentions: Search for StAckA homologs yielded more than 300 non-redundant sequences belonging to the acetokinase family of enzymes that includes acetate, propionate and butyrate kinases. Acetate and propionate kinases share significant sequence identity (~40%) and both groups possess only a low level of identity with butyrate kinases (~20%, Figure 1). Salmonella typhimurium genome codes for three homologs of acetokinase family; namely AckA (acetate kinase), TdcD (propionate kinase) and PduW (putative acetate/propionate kinase). StAckA shares sequence identities of 41% and 40% with StTdcD and StPduW, respectively. Based on sequence analysis, an enzyme corresponding to PduW of S. typhimurium could not be identified in the closely related E. coli. In both S. typhimurium LT2 and E. coli K12, a gene corresponding to a butyrate kinase could not be detected, suggesting differences in utilization of SCFA in microorganisms.


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)

Sequence analysis of acetokinase family of enzymes.  Multiple sequence alignment of St AckA with known structures belonging to acetokinase family. Sequence code: StAckA, S. typhimurium AckA; TmAckA, Thermotoga maritima AckA; MtAckA, Methanosarcina thermophila AckA; MaAckA, Mycobacterium avium AckA; FtTdcD, Francisella tularensis putative acetate/propionate kinase; StTdcD, S. typhimurium propionate kinase; TmBuk2, Thermotoga maritima butyrate kinase 2. All sequences are numbered at the beginning of each block of aligned sequences. StAckA numbering is indicated by every 10 residues using a dot symbol on top of the alignment. Secondary structures of Form-I StAckA and TmBuk2 (PDB:1SAZ) aligned onto their respective sequences are also shown (refer Figure 3A for secondary structure labeling scheme). Colour code: strictly conserved residues are shown in yellow with black background; highly similar regions are shown in blue with grey background. Putative acetate and nucleotide binding residues are marked with orange rhombi and magenta circles, respectively. Residues interacting with citrate in Form-II StAckA structure are highlighted by green triangles.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Sequence analysis of acetokinase family of enzymes. Multiple sequence alignment of St AckA with known structures belonging to acetokinase family. Sequence code: StAckA, S. typhimurium AckA; TmAckA, Thermotoga maritima AckA; MtAckA, Methanosarcina thermophila AckA; MaAckA, Mycobacterium avium AckA; FtTdcD, Francisella tularensis putative acetate/propionate kinase; StTdcD, S. typhimurium propionate kinase; TmBuk2, Thermotoga maritima butyrate kinase 2. All sequences are numbered at the beginning of each block of aligned sequences. StAckA numbering is indicated by every 10 residues using a dot symbol on top of the alignment. Secondary structures of Form-I StAckA and TmBuk2 (PDB:1SAZ) aligned onto their respective sequences are also shown (refer Figure 3A for secondary structure labeling scheme). Colour code: strictly conserved residues are shown in yellow with black background; highly similar regions are shown in blue with grey background. Putative acetate and nucleotide binding residues are marked with orange rhombi and magenta circles, respectively. Residues interacting with citrate in Form-II StAckA structure are highlighted by green triangles.
Mentions: Search for StAckA homologs yielded more than 300 non-redundant sequences belonging to the acetokinase family of enzymes that includes acetate, propionate and butyrate kinases. Acetate and propionate kinases share significant sequence identity (~40%) and both groups possess only a low level of identity with butyrate kinases (~20%, Figure 1). Salmonella typhimurium genome codes for three homologs of acetokinase family; namely AckA (acetate kinase), TdcD (propionate kinase) and PduW (putative acetate/propionate kinase). StAckA shares sequence identities of 41% and 40% with StTdcD and StPduW, respectively. Based on sequence analysis, an enzyme corresponding to PduW of S. typhimurium could not be identified in the closely related E. coli. In both S. typhimurium LT2 and E. coli K12, a gene corresponding to a butyrate kinase could not be detected, suggesting differences in utilization of SCFA in microorganisms.

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