Conservation of structure and activity in Plasmodium purine nucleoside phosphorylases

Abstract

Background: Purine nucleoside phosphorylase (PNP) is central to purine salvage mechanisms in Plasmodium parasites, the causative agents of malaria. Most human malaria results from infection either by Plasmodium falciparum (Pf), the deadliest form of the parasite, or by the widespread Plasmodium vivax (Pv). Whereas the PNP enzyme from Pf has previously been studied in detail, despite the prevalence of Pv little is known about many of the key metabolic enzymes from this parasite, including PvPNP.

Results: The crystal structure of PvPNP is described and is seen to have many features in common with the previously reported structure of PfPNP. In particular, the composition and conformations of the active site regions are virtually identical. The crystal structure of a complex of PfPNP co-crystallised with inosine and arsenate is also described, and is found to contain a mixture of products and reactants - hypoxanthine, ribose and arsenate. The ribose C1' in this hybrid complex lies close to the expected point of symmetry along the PNP reaction coordinate, consistent with a conformation between the transition and product states. These two Plasmodium PNP structures confirm the similarity of structure and mechanism of these enzymes, which are also confirmed in enzyme kinetic assays using an array of substrates. These reveal an unusual form of substrate activation by 2'-deoxyinosine of PvPNP, but not PfPNP.

Conclusion: The close similarity of the Pf and Pv PNP structures allows characteristic features to be identified that differentiate the Apicomplexa PNPs from the human host enzyme. This similarity also suggests there should be a high level of cross-reactivity for compounds designed to inhibit either of these molecular targets. However, despite these similarities, there are also small differences in the activities of the two Plasmodium enzymes.

Figure 1

Catalysis by PNP. Schematic diagrams showing the chemical reactions catalysed by PNP: (a) phosphorolysis, and (b) arsenolysis. These diagrams are based on arsenolytic/hydrolysis transition-state structures of PNPs [17,3,4]. Note protonation at N7, leading to formation of a positive charge, and glycosidic-bond cleavage resulting in formation of a ribo-oxocarbenium ion and negative charge in the purine ring at the transition state. This process occurs prior to nucleophilic attack as the catalytic reaction of PNP follows a SN1 type mechanism. (c) shows the transition state mimc inhibitor, Immucillin H (ImmH).

Figure 2

Overall structure of PvPNP. The ribbon diagram shows a monomer of PvPNP with the secondary structure elements labelled. The bottom panel shows the assembled hexamer with each subunit in a different colour, viewed perpendicular to the three-fold axis.

Figure 3

The active site of the PvPNP. The top panel illustrates the dimer pair and their binding sites with the bound sulphate anions (from the crystal structure) and the inosine substrates (modelled into the PvPNP structure based on superimposition of the PfPNP-inosine structure, PDB:2BSX). The bottom schematic diagram shows possible key residues of the PvPNP active site (numbered according to the system previously used for PfPNP) with hydrogen bonds as dashed lines. Most residues are from the parent monomer, while those labelled 'b' are from the neighbouring subunit across the dimeric surface.

Figure 4

Structure of Pf PNP-HRA complex. (a) active site of typical subunit (b) active site of subunit F (c) corresponding electron density (2F_obs-F_calc, contoured at 1 sigma) for the 'in' and 'out' modes of Arg 27 respectively. Hydrogen bonds are shown as blue dashed lines with distances in Ångstroms, and bound water molecules are shown as red spheres.

Figure 5

Movement of the C1' of the ribose ring throughout catalysis by pPNP. Top figure shows two perpendicular views of an overlay of the ribose and surrounding atoms in PfPNP-inosine/PfPNP-SO₄ (Michaelis complex, green), PfPNP-ImmH (transition state, purple) and PfPNP-hypoxanthine-ribose-AsO₄ complex (post-transition state, yellow) structures. Bottom panel show the distances between C1'-N9 and C1'-nucleophilic oxygen when comparing ligands in different states with the sum of the reaction coordinate distance (Σ) shown. The numbers in brackets are the values proposed by KIE calculations between the Michaelis complex and transition state [17] or those observed in bPNP crystal structures representing the transition state and post-transition state [5]. This figure is similar to those compiled previously for other PNP combinations such as in [5,26,27].

Figure 6

Catalytic mechanism of Plasmodium PNP. Schematic diagram showing the proposed generic Plasmodium PNP reaction mechanism, which is based on that initially proposed for EcPNP [19]. Note that Asp 206 must be in its acidic form prior to protonation. The dotted lines indicate electrostatic interactions, dashed lines are hydrogen bonds and 'w' indicates water molecules. Panel (1) is the binding state, (2) is the pre-catalytic state, (3) is the intermediate state, and (4) is the pre-leaving state. See text for details of each step.

Figure 7

Substrate placement and binding pocket in the Plasmodium and human PNP enzymes. The schematic diagrams represent the human and parasite forms of the enzyme at the catalytic state. The diagram for Plasmodium parasite enzymes (left) is based on the arsenolytic-intermediate-state PfPNP and the sulphate-bound PvPNP structures, with the residues numbered according to the P. falciparum enzyme (+1 for PvPNP), while that for human PNP (right) is based on the transition-state-analogue complexes (PDB ID: 1RR6, [6]) and refined atomic coordinates [20]. Amino acids lining the active sites are represented by spheres, coloured grey for non-polar, green for uncharged polar, blue for positively charged and red for negatively charged amino acids with bound water molecules shown as dark blue spheres. Note residues labelled 'a' are from the parent subunit, while those labelled 'b' from the neighbouring subunit in a dimer pair.

Figure 8

Fitting of initial velocities to different kinetic treatment methods for catalysis of 2'-deoxyinosine by PvPNP. The left (A) shows that application of the substrate activation equation (equation 2 in the text), v = ((V_max1·[S]) + (V_max2·[S]²/K_M2))/(K_M1 + [S] + ([S]²/K_M2)), (-) matches the experimental data better than does the theoretical curve from the substrate inhibition equation (---). The reciprocal plot, 1/v versus 1/[S], with ordinates divided by a factor of 500 (right, B) illustrate the upward curvature at high substrate concentration range, which is similar to an example of a substrate activation case given by [30].