Figure 1.
Purine salvage in P. falciparum.
The enzymes that comprise the purine salvage pathway in Plasmodium: ADA, adenosine deaminase; PNP, purine nucleoside phosphorylase; HXGPRT, hypoxanthine-xanthine-guanine phosphoribosyltransferase. Substrates are: MTA, 5′methylthioadenosine; Ado, adenosine; MTI, 5′methylthioinosine; Ino, inosine; Hyp, hypoxanthine; IMP, inosine monophosphate; AMP, adenosine monophosphate; XMP, xanthine monophosphate; GMP, guanosine monophosphate; ATP, adenosine triphosphate; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine.
Figure 2.
PfPNP substrates and inhibitors.
Structures of substrates (inosine and 5′-methylthioinosine) and immucillin transition state analogues (ImmH and MT-ImmH) of PfPNP utilized for this study.
Figure 3.
Alignment of apicomplexan PNPs.
ClustalW alignment of PNP protein sequences from T. gondii (TgPNP), P.yoelli (PyPNP), and P. falciparium PNP (PfPNP). Residues involved in substrate binding are highlighted [38]. Residues in blue font indicate those surrounding the catalytic domain that were mutated in this study. Amino acids marked: (*) are from the adjacent subunit, (∧) residues are associated with the hydrophobic cavity for accepting the 5′-Methylthio group of MTI.
Table 1.
Kinetic constants for mutant and wild type PNPs from P. falciparum and T. gondii.
Table 2.
Kinetic constants for P. falciparum PNP mutants simulating T. gondii PNP.
Table 3.
Inhibition constants for Immucillins.
Figure 4.
Catalytic Site of the V66I:V73I:Y160F PfPNP mutant.
Cross-eyed stereo view of catalytic site of the triple mutant (V66I:V73I:Y160F) PfPNP showing bound ligand, ImmH, in a 2Fo-Fc map (blue) contoured at 1.0ó. The resolution for this map is 2.8 Å. The figure was prepared with MacPyMol [23].
Figure 5.
Structure of the V66I:V73I:Y160F PfPNP mutant with transition state inhibitor ImmH.
A) Cross-eyed stereo views of the catalytic site contacts in V66I:V73I:Y160F PfPNP with the transition state analogue inhibitor ImmH and PO43−. The figure was created using MacPyMol [23]. Light blue side chains show the parental monomer surrounding the bound ImmH (green), while the yellow side chains indicate residues contributed from the adjacent subunit. The highlighted imino nitrogen is in blue and the 5′-hydroxyl oxygen is in red. B) Side by side images of the decreased convalent interactions located in the enzymatic pocket of V66I:V73I:Y160F PfPNP mutant (3FOW) on the right with WT PfPNP (1NW4) as a comparison.
Figure 6.
Catalytic site contacts for ImmH and PO43− at the active site of V66I:V73I:Y160F PfPNP.
The schematic shows the catalytic site of the triple mutant Val66Ile:Val73Ile:Tyr160Phe PfPNP with ImmH and phosphate in the active site, which is at the interface of 2 subunits within the hexameric structure (a trimer of dimers). Amino acids are from the parent subunit unless labeled with b, which marks residues from the adjacent subunit. Dashed lines indicate hydrogen bonding. Distances are shown in Angstroms.
Figure 7.
Cross-eyed stereo views of the superposition of Immucillins bound in the catalytic site of V66I:V73I:Y160F PfPNP.
A) V66I:V73I:Y160F PfPNP with ImmH (PDB ID: 3FOW) (blue) overlays Wild type PfPNP with ImmH (PDB ID: 1NW4) in orange. B) An isolated overlay of V66I:V73I:Y160F PfPNP: ImmH (orange) and Wildtype PfPNP: MT-ImmH (1Q1G) (purple). The panel shows side chains in surrounding complex with MT-ImmH in the active pocket shifted relative to ImmH.
Table 4.
V66I:V73I:Y160F PfPNP Mutant Residues R-Group Atom Distances to Immucillins.