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Mechanism of hydrolysis of phosphate esters by the dimetal center of 5′-nucleotidase based on crystal structures1

https://doi.org/10.1006/jmbi.2001.4656Get rights and content

Abstract

5′-Nucleotidase belongs to a large superfamily of distantly related dinuclear metallophosphatases including the Ser/Thr protein phosphatases and purple acid phosphatases. The protein undergoes a 96° domain rotation between an open (inactive) and a closed (active) enzyme form. Complex structures of the closed form with the products adenosine and phosphate, and with the substrate analogue inhibitor α,β-methylene ADP, have been determined at 2.1 Å and 1.85 Å resolution, respectively. In addition, a complex of the open form of 5′-nucleotidase with ATP was analyzed at a resolution of 1.7 Å. These structures show that the adenosine group binds to a specific binding pocket of the C-terminal domain. The adenine ring is stacked between Phe429 and Phe498. The N-terminal domain provides the ligands to the dimetal cluster and the conserved His117, which together form the catalytic core structure. However, the three C-terminal arginine residues 375, 379 and 410, which are involved in substrate binding, may also play a role in transition-state stabilization. The β-phosphate group of the inhibitor is terminally coordinated to the site 2 metal ion. The site 1 metal ion coordinates a water molecule which is in an ideal position for a nucleophilic attack on the phosphorus atom, assuming an in-line mechanism of phosphoryl transfer. Another water molecule bridges the two metal ions.

Introduction

Several phosphatases and phosphotransferases utilize a two-metal-ion mechanism to catalyze a phosphoryl transfer reaction.1, 2 The metal ions are usually in close proximity (3-4 Å) and both participate directly in the catalytic steps. 5′-Nucleotidase (5′-NT) is a member of a large superfamily of metallophosphatases which contain such a dinuclear metal center at the C-terminal end of two sandwiched βαβαβ-motifs. This superfamily comprises phosphatases with remarkably diverse substrate specificities, including protein phosphatases, nucleotidases, and nucleases. Structures have been determined for purple acid phosphatase (PAP) from kidney beans,3, 4 from rat5, 6 and from pig;7 for the serine/threonine protein phosphatases 1 (PP-1)8, 9 and 2B (PP-2B),10, 11 also known as calcineurin; and for Escherichia coli 5′-NT.12 Alignments revealed two signature motifs, motif A, DXH(X)∼25 GDXXD(X)∼25GNH[D/E],13, 14 and a shorter second motif B, GH-(X)∼50-GHX[H/X],4 which contain most of the conserved metal-coordinating residues.

E. coli 5′-NT is a zinc-containing enzyme which displays UDP-sugar hydrolase (E.C. 3.6.1.45) as well as 5′-nucleotidase (E.C. 3.1.3.5) activity.15, 16, 17 The enzyme is secreted into the periplasm and can catalyze the degradation of external UDP-glucose to uridine, glucose 1-phosphate and phosphate for utilization by the cell. Together with α-d-sugar-1-phosphatase, it presents a complete system for the degradation of UDP-sugars to uridine and non-phosphorylated sugars which can readily penetrate the cell membrane.15 Purification of 5′-NT from E. coli grown in the presence of 65Zn demonstrated that the enzyme contains zinc. It is, however, not clear if both metal binding sites of E. coli 5′-NT are occupied by zinc in vivo or if the enzyme contains a heteronuclear metal cluster.

The bacterial 5′-NTs show low but significant sequence homology to their animal counterparts, indicative of a common evolutionary origin and similar structures.18 The surface-located animal 5′-NTs play a role in the hydrolysis of extracellular nucleotides, such as ATP, ADP, UTP or diadenosine polyphosphates, which serve as extracellular messengers.19, 20 The hydrolysis of the nucleotides is catalyzed by membrane-bound extracellular nucleotidases (ectonucleotidases), including ATPases, apyrases, and 5′-nucleotidases, which dephosphorylate ATP to ADP, AMP, and adenosine. Besides their role in the cascade that hydrolyses ATP and produces adenosine, surface-located 5′-NTs, which are glycosylated and sialylated, have also been implicated in cell-matrix and cell-cell interactions and in transmembrane signalling. In the case of human ecto-5′-nucleotidase (CD73), it has been shown that these functions are independent of the nucleotidase activity.21

We have previously determined the structure of 5′-NT from E. coli.12 The enzyme consists of two domains which are linked by an α-helix of 19 residues. The catalytic dimetal center is located at the interface between the two domains. 5′-NT exists in an inactive (open) and an active (closed) conformation, which are related by a domain rotation of 96°.22 The structure of open conformation showed that the ligands to the dimetal center and a catalytic Asp-His pair are part of the N-terminal domain. On the basis of this structure it appeared as if no residues of the C-terminal domain are positioned to participate in the catalytic steps.

Here, we analyze the open and closed forms of the enzyme in complex with substrates or substrate analogues and with the reaction products, adenosine and phosphate. These structures reveal the substrate specificity pocket and additional residues of the C-terminal domain which are involved in substrate binding and possibly in the catalytic steps. The structure of the closed conformation with a substrate analogue provides the first structural data on the substrate binding mode for any enzyme of the dinuclear metallophosphatase superfamily. These data are analyzed and discussed in relation to the enzyme mechanism.

Section snippets

Results

We have obtained four different crystal forms of E. coli 5′-NT that are suitable for diffraction studies. Crystal forms I and II contain open enzyme forms, whereas crystals III and IV contain closed enzyme forms.22 Here, we analyze the structure of ATP bound to 5′-NT in crystal form I, which contains one molecule in the asymmetric unit (structure I-ATP), the structure of a 5′-NT/adenosine/PO4-complex in crystal form III, which contains two independent molecules (designated A and B), and the

Perturbance of the substrate binding site resulting from the domain rotation

The substrate binding site of 5′-NT is located in a cleft between the two protein domains. Residues from both domains participate in binding the substrate Figure 2, Figure 4. It is clear from the structure of the open conformation of 5′-NT with ATP, that the 96° domain rotation destroys the catalytic pocket and that the open conformation most likely cannot hydrolyze nucleotides. The four closed conformations observed in the complex of 5′-NT with AMPCP also differ in the interdomain rotation

Crystallization and inhibitor soaking

Three crystal forms have been used in this study. Details of the crystallization are described in the accompanying paper.22 The buffers in which the crystals were incubated prior to data collection are as follows. Crystal form I (space group P41212): 60 % saturated Li2SO4, 0.1 M sodium acetate (pH 6.6) and 0.3 M CsCl; crystal form III (P212121): 100 mM sodium citrate (pH 6.0), 80 mM LiCl, 5 mM KH2PO4, 5 mM adenosine, 10 mM MnCl2, 16 % (w/v) PEG monomethylether 5000 and 25 % (v/v) ethylenediol;

Acknowledgements

We thank W. Saenger for generous support. P. Tucker, E. Pohl, and W. Rypniewski are acknowledged for support with the data collection at the EMBL beamlines at DESY, Hamburg. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to N. S.

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