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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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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.
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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.

Mentions: As the composition of the active sites is identical between the PvPNP and PfPNP structures, it appears reasonable to conclude that both enzymes use the same residues and have the same catalytic mechanism. Fusing a multitude of previous mechanistic studies of other PNPs with the various Plasmodium PNP crystal structures enables a generic mechanism for the Plasmodium enzymes to be summarised (Figure 6). This differs from the mammalian (trimeric) PNP mechanism primarily in the identity and contributions of several of the key catalytic amino acids. These include the anion binding site which is formed from three arginine residues in pPNPs, whereas from one arginine and one histidine in hPNP; the proton-donating residue is Asp 206 in pPNP and Asn 243 in hPNP; residues in the ribose binding pocket also differ particularly a charged Glu 184 in pPNP relative to the hydrophobic Tyr 88 in hPNP, and in pPNP there are cavities adjacent to the O5' of the ribose and N1 and C2 of the base, which are filled by hydrophobic residues for the former and Glu 201 for the latter in hPNP (Figure 7). These accumulated differences suggest that selectivity for the Plasmodium forms should be achievable in the design of PNP inhibitors. This has already been shown with the ImmH series of inhibitors where the derivative MT-ImmH has been reported to bind to PfPNP over 100 fold tighter than to human PNP [6].


Conservation of structure and activity in Plasmodium purine nucleoside phosphorylases.

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

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

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

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.
Mentions: As the composition of the active sites is identical between the PvPNP and PfPNP structures, it appears reasonable to conclude that both enzymes use the same residues and have the same catalytic mechanism. Fusing a multitude of previous mechanistic studies of other PNPs with the various Plasmodium PNP crystal structures enables a generic mechanism for the Plasmodium enzymes to be summarised (Figure 6). This differs from the mammalian (trimeric) PNP mechanism primarily in the identity and contributions of several of the key catalytic amino acids. These include the anion binding site which is formed from three arginine residues in pPNPs, whereas from one arginine and one histidine in hPNP; the proton-donating residue is Asp 206 in pPNP and Asn 243 in hPNP; residues in the ribose binding pocket also differ particularly a charged Glu 184 in pPNP relative to the hydrophobic Tyr 88 in hPNP, and in pPNP there are cavities adjacent to the O5' of the ribose and N1 and C2 of the base, which are filled by hydrophobic residues for the former and Glu 201 for the latter in hPNP (Figure 7). These accumulated differences suggest that selectivity for the Plasmodium forms should be achievable in the design of PNP inhibitors. This has already been shown with the ImmH series of inhibitors where the derivative MT-ImmH has been reported to bind to PfPNP over 100 fold tighter than to human PNP [6].

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