Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Biochemical characterization of the class B acid phosphatase (AphA) of Escherichia coli MG1655
Introduction
The Escherichia coli AphA protein (AphA_ECOLI, swissprot accession: P32697) is a molecular class B bacterial phosphatase that belongs to the DDDD superfamily of phosphohydrolases [1]. AphA is an oligomeric protein made of four identical 25-kDa subunits that is secreted in the periplasmic space. The enzyme exhibits optimal activity at acidic pH values and requires a metal co-factor for activity, as suggested by susceptibility to inhibition by EDTA [2] and by crystallographic data [3].
Preliminary functional characterization showed that the enzyme is able to dephosphorylate various organic phosphomonoesters, including 5′- and 3′-mononucleotides, 2′-deoxy-5′-mononucleotides, aryl-phosphates and glycerol 2-phosphate, being also able to catalyze the transfer of phosphate from p-nitrophenyl phosphate (pNPP) to hydroxyl groups of other organic compounds [2].
The crystal structure of the AphA enzyme has been recently solved [3], revealing that, despite the lack of sequence homology, AphA exhibits a haloacid dehalogenase fold similar to that of other phosphatases such as the phosphoserine phosphatase (PSP) of Methanococcus jannaschii (PDB: 1F5S) [3], [4], and the human mitochondrial 5′-(3′)-deoxyribonucleotidase (dNT-2) (PDB: 1MH9) [3], [5].
In this work, we report a biochemical characterization of the AphA enzyme and discuss some functional features of the enzyme in relation to the protein three-dimensional structure.
Section snippets
AphA production and purification
The AphA enzyme was produced in E. coli DH5α (pATac) essentially as described previously [6]. The enzyme was extracted from cells grown aerobically at 37 °C in Super Broth [7] supplemented with 45 mM potassium phosphate buffer pH 7.2 and ampicillin (250 μg/ml) for plasmid selection. The culture was induced with isopropyl-β-d-thiogalactopyranoside (IPTG) (0.5 mM final concentration) when A600 reached a value of 0.5. Cells were collected by centrifugation (15,000 × g for 45 min at 4 °C) 15 h after
Kinetic parameters of AphA with organic phosphoesters
Kinetic parameters of AphA (KM and kcat) were determined with several organic phosphoesters under initial rate conditions. Results of these experiments confirmed that AphA is active on several phosphomonoesters, but not on 3′,5′-cAMP, ADP or ATP. Overall, the 3′-mononucleotides and 3′-monodeoxynucleotides appeared to be the best substrates (kcat/KM ratios >107 M−1 × s−1). Also the 5′-mononucleotides and 5′-monodeoxynucleotides appeared to behave as very good substrates (kcat/KM ratios >106 M−1 × s−1
Discussion
Kinetic analysis of the AphA enzyme indicated that, although capable of dephosphorylating several different phosphomonoesters, it clearly exhibits a strong preference for 3′- and (to a somewhat lower extent) for 5′-mononucleotides and monodeoxynucleotides. AphA, therefore can be considered a broad-spectrum nucleotidase highly active against both 3′- and 5′-mononucleotides and monodeoxynucleotides. These findings would suggest that AphA could play a role in scavenging nucleotides that enter the
Acknowledgments
This work was supported in part by a grant (PAR “Servizi”) from University of Siena. We would like to thank Jean-Denis Docquier for helpful discussions and advice in determining kinetic parameters of the AphA enzyme.
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Equally contributed to this work.