Abstract
The AphA enzyme of Escherichia coli, a molecular class B periplasmic phosphatase that belongs to the DDDD superfamily of phosphohydrolases, was purified and subjected to biochemical characterization. Kinetic analysis with several substrates revealed that the enzyme essentially behaves as a broad-spectrum nucleotidase highly active on 3′- and 5′- mononucleotides and monodeoxynucleotides, but not active on cyclic nucleotides, or nucleotides di- and triphosphate. Mononucleotides are degraded to nucleosides, and AphA apparently does not exhibit any nucleotide phosphomutase activity. However, it can transphosphorylate nucleosides in the presence of phosphate donors. Kinetic properties of AphA are consistent with structural data, and suggest a role for the hydrophobic pocket present in the active site crevice, made by residues Phe 56, Leu71, Trp77 and Tyr193, in conferring preferential substrate specificity by accommodating compounds with aromatic rings. AphA was inhibited by several chelating agents, including EDTA, EGTA, 1,10-phenanthroline and dipicolinic acid, with EDTA being apparently the most powerful inhibitor.
| Original language | English |
|---|---|
| Pages (from-to) | 13-19 |
| Number of pages | 7 |
| Journal | Biochimica et Biophysica Acta - Proteins and Proteomics |
| Volume | 1764 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - Jan 2006 |
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Keywords
- AphA
- Biochemical characterization
- class B acid phosphatase
- Escherichia coli
- Kinetic analysis
ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Genetics
Cite this
Biochemical characterization of the class B acid phosphatase (AphA) of Escherichia coli MG1655. / Passariello, Claudio; Forleo, Costantino; Micheli, Vanna; Schippa, Serena; Leone, Rosalida; Mangani, Stefano; Thaller, Maria Cristina; Rossolini, Gian Maria.
In: Biochimica et Biophysica Acta - Proteins and Proteomics, Vol. 1764, No. 1, 01.2006, p. 13-19.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Biochemical characterization of the class B acid phosphatase (AphA) of Escherichia coli MG1655
AU - Passariello, Claudio
AU - Forleo, Costantino
AU - Micheli, Vanna
AU - Schippa, Serena
AU - Leone, Rosalida
AU - Mangani, Stefano
AU - Thaller, Maria Cristina
AU - Rossolini, Gian Maria
PY - 2006/1
Y1 - 2006/1
N2 - The AphA enzyme of Escherichia coli, a molecular class B periplasmic phosphatase that belongs to the DDDD superfamily of phosphohydrolases, was purified and subjected to biochemical characterization. Kinetic analysis with several substrates revealed that the enzyme essentially behaves as a broad-spectrum nucleotidase highly active on 3′- and 5′- mononucleotides and monodeoxynucleotides, but not active on cyclic nucleotides, or nucleotides di- and triphosphate. Mononucleotides are degraded to nucleosides, and AphA apparently does not exhibit any nucleotide phosphomutase activity. However, it can transphosphorylate nucleosides in the presence of phosphate donors. Kinetic properties of AphA are consistent with structural data, and suggest a role for the hydrophobic pocket present in the active site crevice, made by residues Phe 56, Leu71, Trp77 and Tyr193, in conferring preferential substrate specificity by accommodating compounds with aromatic rings. AphA was inhibited by several chelating agents, including EDTA, EGTA, 1,10-phenanthroline and dipicolinic acid, with EDTA being apparently the most powerful inhibitor.
AB - The AphA enzyme of Escherichia coli, a molecular class B periplasmic phosphatase that belongs to the DDDD superfamily of phosphohydrolases, was purified and subjected to biochemical characterization. Kinetic analysis with several substrates revealed that the enzyme essentially behaves as a broad-spectrum nucleotidase highly active on 3′- and 5′- mononucleotides and monodeoxynucleotides, but not active on cyclic nucleotides, or nucleotides di- and triphosphate. Mononucleotides are degraded to nucleosides, and AphA apparently does not exhibit any nucleotide phosphomutase activity. However, it can transphosphorylate nucleosides in the presence of phosphate donors. Kinetic properties of AphA are consistent with structural data, and suggest a role for the hydrophobic pocket present in the active site crevice, made by residues Phe 56, Leu71, Trp77 and Tyr193, in conferring preferential substrate specificity by accommodating compounds with aromatic rings. AphA was inhibited by several chelating agents, including EDTA, EGTA, 1,10-phenanthroline and dipicolinic acid, with EDTA being apparently the most powerful inhibitor.
KW - AphA
KW - Biochemical characterization
KW - class B acid phosphatase
KW - Escherichia coli
KW - Kinetic analysis
UR - http://www.scopus.com/inward/record.url?scp=33644839936&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33644839936&partnerID=8YFLogxK
U2 - 10.1016/j.bbapap.2005.08.028
DO - 10.1016/j.bbapap.2005.08.028
M3 - Article
C2 - 16297670
AN - SCOPUS:33644839936
VL - 1764
SP - 13
EP - 19
JO - Biochimica et Biophysica Acta - Proteins and Proteomics
JF - Biochimica et Biophysica Acta - Proteins and Proteomics
SN - 1570-9639
IS - 1
ER -