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Analysis of proteins with the 'hot dog' fold: prediction of function and identification of catalytic residues of hypothetical proteins.

Pidugu LS, Maity K, Ramaswamy K, Surolia N, Suguna K - BMC Struct. Biol. (2009)

Bottom Line: The hot dog fold has been found in more than sixty proteins since the first report of its existence about a decade ago.This study reveals that though the basic architecture of the fold is well conserved in these proteins, significant differences exist in their sequence, nature of substrate and oligomerization.The analysis led to predictions regarding the functional classification and identification of possible catalytic residues of a number of hot dog fold-containing hypothetical proteins whose structures were determined in high throughput structural genomics projects.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India. plsmukhi@gmail.com

ABSTRACT

Background: The hot dog fold has been found in more than sixty proteins since the first report of its existence about a decade ago. The fold appears to have a strong association with fatty acid biosynthesis, its regulation and metabolism, as the proteins with this fold are predominantly coenzyme A-binding enzymes with a variety of substrates located at their active sites.

Results: We have analyzed the structural features and sequences of proteins having the hot dog fold. This study reveals that though the basic architecture of the fold is well conserved in these proteins, significant differences exist in their sequence, nature of substrate and oligomerization. Segments with certain conserved sequence motifs seem to play crucial structural and functional roles in various classes of these proteins.

Conclusion: The analysis led to predictions regarding the functional classification and identification of possible catalytic residues of a number of hot dog fold-containing hypothetical proteins whose structures were determined in high throughput structural genomics projects.

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TB tetramer of E. coli PaaI. The kinked conformation of the N-terminal helices is indicated by arrows (PDB code: 2FS2).
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Figure 4: TB tetramer of E. coli PaaI. The kinked conformation of the N-terminal helices is indicated by arrows (PDB code: 2FS2).

Mentions: PaaI is a thioesterase involved in the phenylacetic acid catabolic pathway, catalyzing the degradation of phenylacetyl-CoA [22]. PaaI from Thermus thermophilus (TtPaaI; PDB code 1J1Y) and E. coli (EcPaaI: PDB code: 2FS2) have been characterized both structurally and biochemically. They also form TB tetramers similar to the 4HBT-II subfamily. A novel induced fit mechanism was observed in the case of TtPaaI, where only two of the four active sites accommodate the ligand, indicating half of the sites reactivity. A rotation of up to 2° of the subunits upon ligand binding introduces a negative cooperativity preventing the binding of ligands at the other two active sites [23]. In the case of EcPaaI, the overall structure is similar to that of TtPaaI except for the presence of an additional long N-terminal helix that lines the active site (Fig. 4).The helix has a kink in two subunits of the tetramer [24] giving rise to two different catalytic platforms. It was further confirmed by site directed mutagenesis of the active site residues in both conformations of the helix that the subunit with the kinked N-terminal helix is catalytically active [24]. Four hypothetical proteins (PDB codes: 1ZKI from P. aeruginosa, 2CY9 from M. musculus, 2QWZ from Silicibacter sp., 2PIM from R. eutropha) and a putative human enzyme (2F0X[25]) form tetramers of the type TB similar to that of TtPaaI whereas another protein (3BBJ) is a double hot dog with a DdhB type of quaternary structure. Hypothetical proteins 1IXL (P. horikoshii) [26], 2HBO (C. crescentus), 2PRX (S. loihica) and 2OV9 (Rhodococcus sp.) possess the active site consensus sequence motif (see Additional file 2) of PaaI but show variation in the consensus sequence motif located at the tetramer interface (see Additional file 2). These four proteins form dimers of hot dogs. In TtPaaI, Asp48 identified as the catalytic residue [23], occurs in the consensus sequence motif and is found to be conserved in all the proteins of this subfamily. The variation in the second consensus sequence motif that is present at the tetramer interfaces, might be responsible for the proteins of this subfamily having different oligomeric structures. However, the substrate specificities of these proteins are yet to be confirmed. The structure of 2OV9 is different from the others with an additional long loop consisting of two helices from each subunit forming a four-helix bundle as a separate domain (Fig. 5).


Analysis of proteins with the 'hot dog' fold: prediction of function and identification of catalytic residues of hypothetical proteins.

Pidugu LS, Maity K, Ramaswamy K, Surolia N, Suguna K - BMC Struct. Biol. (2009)

TB tetramer of E. coli PaaI. The kinked conformation of the N-terminal helices is indicated by arrows (PDB code: 2FS2).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2698920&req=5

Figure 4: TB tetramer of E. coli PaaI. The kinked conformation of the N-terminal helices is indicated by arrows (PDB code: 2FS2).
Mentions: PaaI is a thioesterase involved in the phenylacetic acid catabolic pathway, catalyzing the degradation of phenylacetyl-CoA [22]. PaaI from Thermus thermophilus (TtPaaI; PDB code 1J1Y) and E. coli (EcPaaI: PDB code: 2FS2) have been characterized both structurally and biochemically. They also form TB tetramers similar to the 4HBT-II subfamily. A novel induced fit mechanism was observed in the case of TtPaaI, where only two of the four active sites accommodate the ligand, indicating half of the sites reactivity. A rotation of up to 2° of the subunits upon ligand binding introduces a negative cooperativity preventing the binding of ligands at the other two active sites [23]. In the case of EcPaaI, the overall structure is similar to that of TtPaaI except for the presence of an additional long N-terminal helix that lines the active site (Fig. 4).The helix has a kink in two subunits of the tetramer [24] giving rise to two different catalytic platforms. It was further confirmed by site directed mutagenesis of the active site residues in both conformations of the helix that the subunit with the kinked N-terminal helix is catalytically active [24]. Four hypothetical proteins (PDB codes: 1ZKI from P. aeruginosa, 2CY9 from M. musculus, 2QWZ from Silicibacter sp., 2PIM from R. eutropha) and a putative human enzyme (2F0X[25]) form tetramers of the type TB similar to that of TtPaaI whereas another protein (3BBJ) is a double hot dog with a DdhB type of quaternary structure. Hypothetical proteins 1IXL (P. horikoshii) [26], 2HBO (C. crescentus), 2PRX (S. loihica) and 2OV9 (Rhodococcus sp.) possess the active site consensus sequence motif (see Additional file 2) of PaaI but show variation in the consensus sequence motif located at the tetramer interface (see Additional file 2). These four proteins form dimers of hot dogs. In TtPaaI, Asp48 identified as the catalytic residue [23], occurs in the consensus sequence motif and is found to be conserved in all the proteins of this subfamily. The variation in the second consensus sequence motif that is present at the tetramer interfaces, might be responsible for the proteins of this subfamily having different oligomeric structures. However, the substrate specificities of these proteins are yet to be confirmed. The structure of 2OV9 is different from the others with an additional long loop consisting of two helices from each subunit forming a four-helix bundle as a separate domain (Fig. 5).

Bottom Line: The hot dog fold has been found in more than sixty proteins since the first report of its existence about a decade ago.This study reveals that though the basic architecture of the fold is well conserved in these proteins, significant differences exist in their sequence, nature of substrate and oligomerization.The analysis led to predictions regarding the functional classification and identification of possible catalytic residues of a number of hot dog fold-containing hypothetical proteins whose structures were determined in high throughput structural genomics projects.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India. plsmukhi@gmail.com

ABSTRACT

Background: The hot dog fold has been found in more than sixty proteins since the first report of its existence about a decade ago. The fold appears to have a strong association with fatty acid biosynthesis, its regulation and metabolism, as the proteins with this fold are predominantly coenzyme A-binding enzymes with a variety of substrates located at their active sites.

Results: We have analyzed the structural features and sequences of proteins having the hot dog fold. This study reveals that though the basic architecture of the fold is well conserved in these proteins, significant differences exist in their sequence, nature of substrate and oligomerization. Segments with certain conserved sequence motifs seem to play crucial structural and functional roles in various classes of these proteins.

Conclusion: The analysis led to predictions regarding the functional classification and identification of possible catalytic residues of a number of hot dog fold-containing hypothetical proteins whose structures were determined in high throughput structural genomics projects.

Show MeSH