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Conservation of structure and activity in Plasmodium purine nucleoside phosphorylases.

Chaikuad A, Brady RL - BMC Struct. Biol. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, University of Bristol, Bristol, BS8 1TD, UK. Apirat.Chaikuad@sgc.ox.ac.uk

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.

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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.
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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.

Mentions: The PvPNP crystal structure was refined against 1.85 Å resolution data (summarised in Table 1) and the model contains all of the protein residues with the exception of the disordered active site loop (residues 212–224). The R32 crystals displayed considerable anisotropy in the distribution of their diffraction intensities leading to the rejection of many higher resolution reflections during processing. Nonetheless, about 70% of the processed reflections have intensities with I/(sigma I) greater than 3 in the highest resolution shell, hence the structure has been refined against all available data to 1.85 Å. The crystallographic asymmetric unit contains a monomer of PvPNP which adopts the familiar single-domain fold topology described previously for hexameric PNPs from other species (e.g. [6,9,12,13]). Each PvPNP monomer is comprised of a 10-stranded β-sheet core, which forms the base of the catalytic site, and eight α-helices, which are involved in subunit contacts (labelled in Figure 2). The total number and position of secondary structure elements in PvPNP are comparable to those in PfPNP, although small variations in the number of β-strands and α-helices are evident due to different assignments when comparing PfPNP structures ([9,6] and the PfPNP solved in this study). An extra α-helix in the ordered active site loop of PfPNP is not observed in the PvPNP structure, but may be formed when the PvPNP active site loop is structured.


Conservation of structure and activity in Plasmodium purine nucleoside phosphorylases.

Chaikuad A, Brady RL - BMC Struct. Biol. (2009)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2721837&req=5

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.
Mentions: The PvPNP crystal structure was refined against 1.85 Å resolution data (summarised in Table 1) and the model contains all of the protein residues with the exception of the disordered active site loop (residues 212–224). The R32 crystals displayed considerable anisotropy in the distribution of their diffraction intensities leading to the rejection of many higher resolution reflections during processing. Nonetheless, about 70% of the processed reflections have intensities with I/(sigma I) greater than 3 in the highest resolution shell, hence the structure has been refined against all available data to 1.85 Å. The crystallographic asymmetric unit contains a monomer of PvPNP which adopts the familiar single-domain fold topology described previously for hexameric PNPs from other species (e.g. [6,9,12,13]). Each PvPNP monomer is comprised of a 10-stranded β-sheet core, which forms the base of the catalytic site, and eight α-helices, which are involved in subunit contacts (labelled in Figure 2). The total number and position of secondary structure elements in PvPNP are comparable to those in PfPNP, although small variations in the number of β-strands and α-helices are evident due to different assignments when comparing PfPNP structures ([9,6] and the PfPNP solved in this study). An extra α-helix in the ordered active site loop of PfPNP is not observed in the PvPNP structure, but may be formed when the PvPNP active site loop is structured.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, University of Bristol, Bristol, BS8 1TD, UK. Apirat.Chaikuad@sgc.ox.ac.uk

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.

Show MeSH
Related in: MedlinePlus