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The calcium activated nucleotidases: A diverse family of soluble and membrane associated nucleotide hydrolyzing enzymes.

Smith TM, Kirley TL - Purinergic Signal. (2006)

Bottom Line: In this review, we present an overview of the diverse family of apyrases first described in the blood-feeding arthropods, including the identification and characterization of the soluble and membrane-bound vertebrate enzymes homologous to these arthropod apyrases.We also describe the enzymatic properties and nucleotide specificities of the expressed enzymes, and insights gained into the structure and function of this calcium activated nucleotidase (CAN) family from biophysical, mutagenesis and crystallography studies.The potential therapeutic value of these proteins is also discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular and Metabolic Diseases, Wyeth Research, Cambridge, Massachusetts, 02140, USA, tmsmith@wyeth.com.

ABSTRACT
It has long been known that the salivary glands of hematophagous (blood-feeding) arthropods secrete soluble apyrases, which are potent nucleotide hydrolyzing enzymes capable of hydrolyzing extracellular ATP and ADP, the latter being a major agonist contributing to platelet aggregation. Only recently, however, has the identification of proteins homologous to these apyrases been reported in non-blood-feeding organisms such as rodents and humans. In this review, we present an overview of the diverse family of apyrases first described in the blood-feeding arthropods, including the identification and characterization of the soluble and membrane-bound vertebrate enzymes homologous to these arthropod apyrases. We also describe the enzymatic properties and nucleotide specificities of the expressed enzymes, and insights gained into the structure and function of this calcium activated nucleotidase (CAN) family from biophysical, mutagenesis and crystallography studies. The potential therapeutic value of these proteins is also discussed.

No MeSH data available.


Related in: MedlinePlus

Structure of the human calcium activated nucleotidase in complex with the GMP-CP substrate analog. Ribbon diagrams of the human protein β-propeller structure as viewed along (A) or perpendicular to (B) the central tunnel. The polypeptide is colored from blue at the C-terminus through to red at the C-terminus. The structure reveals an unusual five-bladed β-propeller with five twisted β-sheets, each formed from four antiparallel β-strands. The interface between neighboring blades is predominantly hydrophobic, with residues on the adjacent faces of the β-sheets in van der Waals contact. The Ca2+ ion (green sphere) is located at the middle of the central tunnel. The GMP-CP molecule is shown in a ball-and-stick representation (CPK color scheme). The propeller has an approximate diameter of 44 Å and height of 30 Å. The atomic coordinates are available at the Protein Data Bank (PDB code 1S1D). Reprinted from Cell, Vol. 116, Dai et al., Structure and Protein Design of a Human Platelet Function Inhibitor, pp 649–659, Copyright (2004), with permission from Elsevier.
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Fig3: Structure of the human calcium activated nucleotidase in complex with the GMP-CP substrate analog. Ribbon diagrams of the human protein β-propeller structure as viewed along (A) or perpendicular to (B) the central tunnel. The polypeptide is colored from blue at the C-terminus through to red at the C-terminus. The structure reveals an unusual five-bladed β-propeller with five twisted β-sheets, each formed from four antiparallel β-strands. The interface between neighboring blades is predominantly hydrophobic, with residues on the adjacent faces of the β-sheets in van der Waals contact. The Ca2+ ion (green sphere) is located at the middle of the central tunnel. The GMP-CP molecule is shown in a ball-and-stick representation (CPK color scheme). The propeller has an approximate diameter of 44 Å and height of 30 Å. The atomic coordinates are available at the Protein Data Bank (PDB code 1S1D). Reprinted from Cell, Vol. 116, Dai et al., Structure and Protein Design of a Human Platelet Function Inhibitor, pp 649–659, Copyright (2004), with permission from Elsevier.

Mentions: In addition to performing extensive mutagenesis studies on the human protein, Dai et al. [20] reported the highresolution crystal structures of the enzyme both in the presence and absence of the non-hydrolyzable GDP substrate analog GMP-CP. The structures revealed a novel nucleotide-binding motif comprising a five-blade beta propeller structure, with a single calcium-binding site in the middle of the central tunnel (Figure 3), coordinated by Ser 100, Glu 147, Glu 216, Ser 277, and Glu 328 (numbering based on the soluble version of the apyrase minus the signal peptide sequence). The single bound calcium ion also contacts a water molecule and the carboxylate group of Asp 101. With the exception of a conserved glutamate substitution in the blood-feeding arthropod proteins for Asp 101, the calcium binding residues are invariant in all members (Figure 2). Consistent with this observation, mutagenesis of these amino acids abolished the enzymatic activity. The presence of the substrate analog GMP-CP revealed that the guanine ring is bounded by Trp 165 and Tyr 239. In addition, the phosphate recognition pocket is lined mostly by hydrophilic residues, and site-directed mutagenesis of these amino acids confirmed their roles in the active site of the enzyme [20].Figure 3


The calcium activated nucleotidases: A diverse family of soluble and membrane associated nucleotide hydrolyzing enzymes.

Smith TM, Kirley TL - Purinergic Signal. (2006)

Structure of the human calcium activated nucleotidase in complex with the GMP-CP substrate analog. Ribbon diagrams of the human protein β-propeller structure as viewed along (A) or perpendicular to (B) the central tunnel. The polypeptide is colored from blue at the C-terminus through to red at the C-terminus. The structure reveals an unusual five-bladed β-propeller with five twisted β-sheets, each formed from four antiparallel β-strands. The interface between neighboring blades is predominantly hydrophobic, with residues on the adjacent faces of the β-sheets in van der Waals contact. The Ca2+ ion (green sphere) is located at the middle of the central tunnel. The GMP-CP molecule is shown in a ball-and-stick representation (CPK color scheme). The propeller has an approximate diameter of 44 Å and height of 30 Å. The atomic coordinates are available at the Protein Data Bank (PDB code 1S1D). Reprinted from Cell, Vol. 116, Dai et al., Structure and Protein Design of a Human Platelet Function Inhibitor, pp 649–659, Copyright (2004), with permission from Elsevier.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2254480&req=5

Fig3: Structure of the human calcium activated nucleotidase in complex with the GMP-CP substrate analog. Ribbon diagrams of the human protein β-propeller structure as viewed along (A) or perpendicular to (B) the central tunnel. The polypeptide is colored from blue at the C-terminus through to red at the C-terminus. The structure reveals an unusual five-bladed β-propeller with five twisted β-sheets, each formed from four antiparallel β-strands. The interface between neighboring blades is predominantly hydrophobic, with residues on the adjacent faces of the β-sheets in van der Waals contact. The Ca2+ ion (green sphere) is located at the middle of the central tunnel. The GMP-CP molecule is shown in a ball-and-stick representation (CPK color scheme). The propeller has an approximate diameter of 44 Å and height of 30 Å. The atomic coordinates are available at the Protein Data Bank (PDB code 1S1D). Reprinted from Cell, Vol. 116, Dai et al., Structure and Protein Design of a Human Platelet Function Inhibitor, pp 649–659, Copyright (2004), with permission from Elsevier.
Mentions: In addition to performing extensive mutagenesis studies on the human protein, Dai et al. [20] reported the highresolution crystal structures of the enzyme both in the presence and absence of the non-hydrolyzable GDP substrate analog GMP-CP. The structures revealed a novel nucleotide-binding motif comprising a five-blade beta propeller structure, with a single calcium-binding site in the middle of the central tunnel (Figure 3), coordinated by Ser 100, Glu 147, Glu 216, Ser 277, and Glu 328 (numbering based on the soluble version of the apyrase minus the signal peptide sequence). The single bound calcium ion also contacts a water molecule and the carboxylate group of Asp 101. With the exception of a conserved glutamate substitution in the blood-feeding arthropod proteins for Asp 101, the calcium binding residues are invariant in all members (Figure 2). Consistent with this observation, mutagenesis of these amino acids abolished the enzymatic activity. The presence of the substrate analog GMP-CP revealed that the guanine ring is bounded by Trp 165 and Tyr 239. In addition, the phosphate recognition pocket is lined mostly by hydrophilic residues, and site-directed mutagenesis of these amino acids confirmed their roles in the active site of the enzyme [20].Figure 3

Bottom Line: In this review, we present an overview of the diverse family of apyrases first described in the blood-feeding arthropods, including the identification and characterization of the soluble and membrane-bound vertebrate enzymes homologous to these arthropod apyrases.We also describe the enzymatic properties and nucleotide specificities of the expressed enzymes, and insights gained into the structure and function of this calcium activated nucleotidase (CAN) family from biophysical, mutagenesis and crystallography studies.The potential therapeutic value of these proteins is also discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular and Metabolic Diseases, Wyeth Research, Cambridge, Massachusetts, 02140, USA, tmsmith@wyeth.com.

ABSTRACT
It has long been known that the salivary glands of hematophagous (blood-feeding) arthropods secrete soluble apyrases, which are potent nucleotide hydrolyzing enzymes capable of hydrolyzing extracellular ATP and ADP, the latter being a major agonist contributing to platelet aggregation. Only recently, however, has the identification of proteins homologous to these apyrases been reported in non-blood-feeding organisms such as rodents and humans. In this review, we present an overview of the diverse family of apyrases first described in the blood-feeding arthropods, including the identification and characterization of the soluble and membrane-bound vertebrate enzymes homologous to these arthropod apyrases. We also describe the enzymatic properties and nucleotide specificities of the expressed enzymes, and insights gained into the structure and function of this calcium activated nucleotidase (CAN) family from biophysical, mutagenesis and crystallography studies. The potential therapeutic value of these proteins is also discussed.

No MeSH data available.


Related in: MedlinePlus