Biochemical characterization of the class B acid phosphatase (AphA) of Escherichia coli MG1655

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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.

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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