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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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Structure of Form-I St AckA.  (A) Tertiary structure of St AckA illustrating the N- (domain-I) and C-terminal (domain-II) domains of the enzyme. The core secondary structural elements (βββαβαβα) are shown in green (strands) and cyan (helices), while insertions of subdomains between secondary structural elements are highlighted in magenta (strands) and yellow (helices). Secondary structural elements of the core are numbered αI1, αI2… for α-helices, 3I1, 3I2… for 310 helices, βI1, βI2… for β-strands in domain-I and as αII1, αII2… for α-helices, 3II1, 3II2… for 310 helices, βII1, βII2… for β-strands in domain-II. Insertion domains are indexed similarly and numbered alphabetically. N- and C- termini of the subunit are also marked. (B) Topology diagram of StAckA indicating the ASKHA core fold and the location of the five conserved motifs. Arrows represent β-strands while cylinders represent helices, respectively. The coloring and secondary structure labeling scheme is similar to that of Figure 3A. (C) Dimeric structure of StAckA. The approximate size of a dimeric unit is 84 x 83 x 69 Å3. A-subunit of the dimer is illustrated using the same colouring scheme to that of Figure 3A. Domain-I and -II of the B-subunit are shown in grey and brown, respectively. The dimer is mainly held by interactions between the C-terminal domains (domain-II) of the two subunits, while domain-I of each subunit protrudes out of the body of the dimeric enzyme. The 2-fold axis is highlighted by dotted line. Secondary structures corresponding to the core helices are labeled.
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Figure 3: Structure of Form-I St AckA. (A) Tertiary structure of St AckA illustrating the N- (domain-I) and C-terminal (domain-II) domains of the enzyme. The core secondary structural elements (βββαβαβα) are shown in green (strands) and cyan (helices), while insertions of subdomains between secondary structural elements are highlighted in magenta (strands) and yellow (helices). Secondary structural elements of the core are numbered αI1, αI2… for α-helices, 3I1, 3I2… for 310 helices, βI1, βI2… for β-strands in domain-I and as αII1, αII2… for α-helices, 3II1, 3II2… for 310 helices, βII1, βII2… for β-strands in domain-II. Insertion domains are indexed similarly and numbered alphabetically. N- and C- termini of the subunit are also marked. (B) Topology diagram of StAckA indicating the ASKHA core fold and the location of the five conserved motifs. Arrows represent β-strands while cylinders represent helices, respectively. The coloring and secondary structure labeling scheme is similar to that of Figure 3A. (C) Dimeric structure of StAckA. The approximate size of a dimeric unit is 84 x 83 x 69 Å3. A-subunit of the dimer is illustrated using the same colouring scheme to that of Figure 3A. Domain-I and -II of the B-subunit are shown in grey and brown, respectively. The dimer is mainly held by interactions between the C-terminal domains (domain-II) of the two subunits, while domain-I of each subunit protrudes out of the body of the dimeric enzyme. The 2-fold axis is highlighted by dotted line. Secondary structures corresponding to the core helices are labeled.

Mentions: 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.


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)

Structure of Form-I St AckA.  (A) Tertiary structure of St AckA illustrating the N- (domain-I) and C-terminal (domain-II) domains of the enzyme. The core secondary structural elements (βββαβαβα) are shown in green (strands) and cyan (helices), while insertions of subdomains between secondary structural elements are highlighted in magenta (strands) and yellow (helices). Secondary structural elements of the core are numbered αI1, αI2… for α-helices, 3I1, 3I2… for 310 helices, βI1, βI2… for β-strands in domain-I and as αII1, αII2… for α-helices, 3II1, 3II2… for 310 helices, βII1, βII2… for β-strands in domain-II. Insertion domains are indexed similarly and numbered alphabetically. N- and C- termini of the subunit are also marked. (B) Topology diagram of StAckA indicating the ASKHA core fold and the location of the five conserved motifs. Arrows represent β-strands while cylinders represent helices, respectively. The coloring and secondary structure labeling scheme is similar to that of Figure 3A. (C) Dimeric structure of StAckA. The approximate size of a dimeric unit is 84 x 83 x 69 Å3. A-subunit of the dimer is illustrated using the same colouring scheme to that of Figure 3A. Domain-I and -II of the B-subunit are shown in grey and brown, respectively. The dimer is mainly held by interactions between the C-terminal domains (domain-II) of the two subunits, while domain-I of each subunit protrudes out of the body of the dimeric enzyme. The 2-fold axis is highlighted by dotted line. Secondary structures corresponding to the core helices are labeled.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Structure of Form-I St AckA. (A) Tertiary structure of St AckA illustrating the N- (domain-I) and C-terminal (domain-II) domains of the enzyme. The core secondary structural elements (βββαβαβα) are shown in green (strands) and cyan (helices), while insertions of subdomains between secondary structural elements are highlighted in magenta (strands) and yellow (helices). Secondary structural elements of the core are numbered αI1, αI2… for α-helices, 3I1, 3I2… for 310 helices, βI1, βI2… for β-strands in domain-I and as αII1, αII2… for α-helices, 3II1, 3II2… for 310 helices, βII1, βII2… for β-strands in domain-II. Insertion domains are indexed similarly and numbered alphabetically. N- and C- termini of the subunit are also marked. (B) Topology diagram of StAckA indicating the ASKHA core fold and the location of the five conserved motifs. Arrows represent β-strands while cylinders represent helices, respectively. The coloring and secondary structure labeling scheme is similar to that of Figure 3A. (C) Dimeric structure of StAckA. The approximate size of a dimeric unit is 84 x 83 x 69 Å3. A-subunit of the dimer is illustrated using the same colouring scheme to that of Figure 3A. Domain-I and -II of the B-subunit are shown in grey and brown, respectively. The dimer is mainly held by interactions between the C-terminal domains (domain-II) of the two subunits, while domain-I of each subunit protrudes out of the body of the dimeric enzyme. The 2-fold axis is highlighted by dotted line. Secondary structures corresponding to the core helices are labeled.
Mentions: 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.

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