Crystal structure of Escherichia coli purine nucleoside phosphorylase complexed with acyclovir

Abstract

Escherichia coli purine nucleoside phosphorylase (PNP), which catalyzes the reversible phosphorolysis of purine ribonucleosides, belongs to the family I hexameric PNPs. Owing to their key role in the purine salvage pathway, PNPs are attractive targets for drug design against some pathogens. Acyclovir (ACV) is an acyclic derivative of the PNP substrate guanosine and is used as an antiviral drug for the treatment of some human viral infections. The crystalline complex of E. coli PNP with acyclovir was prepared by co-crystallization in microgravity using counter-diffusion through a gel layer in a capillary. The structure of the E. coli PNP-ACV complex was solved at 2.32 Å resolution using the molecular-replacement method. The ACV molecule is observed in two conformations and sulfate ions were located in both the nucleoside-binding and phosphate-binding pockets of the enzyme. A comparison with the complexes of other hexameric and trimeric PNPs with ACV shows the similarity in acyclovir binding by these enzymes.

Keywords: Escherichia coli; acyclovir; crystal structure; inhibitors; purine nucleoside phosphorylase; structure-based drug design; tumour-directed gene therapy.

Figure 1

Sequence alignment of E. coli, B. subtilis and human PNPs. This figure was created using ESPript (Gouet et al., 2003 ▸).

Figure 2

(a, b) Acyclovir in the active site of the E. coli PNP subunit. The electron-density map was calculated without ligands with coefficients 2|F _o| − |F _c| and is contoured at 2σ for the two conformations of acyclovir. (c) A hexameric molecule of E. coli PNP with an acyclovir molecule and a sulfate ion in the active sites. These figures were created using PyMOL (https://pymol.org/2/).

Figure 3

(a) Active site of E. coli PNP with the acyclovir molecule bound in two conformations and a sulfate ion. One subunit is shown in green and the second subunit is shown in blue. Conformational changes of the apoenzyme upon binding acyclovir and sulfate ion. (b) The superposition on C^α atoms of apo PNP and PNP complexed with ACV and sulfate ion is shown. These figures were created using PyMOL (https://pymol.org/2/).

Figure 4

Comparison of the active sites of several PNPs complexed with different guanosine derivatives. Acyclovir is shown (a) in the active sites of E. coli PNP (green) and B. subtilis PNP (pink) and (b) in the active sites of E. coli PNP (green) and human PNP (pink). (c) ACV in the active site of E. coli PNP (green) and 2′-deoxyguanosine bound in the active site of B. subtilis PNP (blue). (d) ACV (N9-acycloguanosine) in the active site of E. coli PNP (green) and N7-acycloguanosine in the active site of calf spleen PNP (grey). The corresponding structures are superimposed on the C^α atoms of amino acids in the active site. These figures were created using PyMOL (https://pymol.org/2/).