Abstract
Plasmodium falciparum enolase (Pfeno) is a dimeric enzyme with multiple moonlighting functions. This enzyme is thus a potential target for anti‐malarial treatments. A unique feature of Pfeno is the presence of a pentapeptide insert 104 EWGWS 108. The functional role of tryptophan residues in this insert was investigated using site‐directed mutagenesis. Replacement of these two Trp residues with alanines (or lysines) resulted in a near complete loss of enolase activity and dissociation of the normal dimeric form into monomers. Molecular modeling indicated that 340R forms π‐cation bonds with the aromatic rings of 105W and 46Y. Mutation induced changes in the interactions among these three residues were presumably relayed to the inter‐subunit interface via a coil formed by 46Y : 59Y, resulting in the disruption of a salt bridge between 11R : 425E and a π‐cation interaction between 11R : 59Y. This led to a drop of ~ 4 kcal·mole−1 in the inter‐subunit docking energy in the mutant, causing a ~ 103 fold decrease in affinity. Partial restoration of the inter‐subunit interactions led to reformation of dimers and also restored a significant fraction of the lost enzyme activity. These results suggested that the perturbations in the conformation of the surface loop containing the insert sequence were relayed to the interface region, causing dimer dissociation that, in turn, disrupted the enzyme's active site. Since Plasmodium enolase is a moonlighting protein with multiple parasite‐specific functions, it is likely that these functions may map on to the highly conserved unique insert region of this protein.EnzymesEnolase(EC4.2.1.11).
Highlights
Plasmodium falciparum enolase (Pfeno) is a dimeric enzyme with multiple moonlighting functions
Partial restoration of the inter-subunit interactions led to reformation of dimers and restored a significant fraction of the lost enzyme activity. These results suggested that the perturbations in the conformation of the surface loop containing the insert sequence were relayed to the interface region, causing dimer dissociation that, in turn, disrupted the enzyme’s active site
The parasite specific insert sequence 104EWGWS108 is a part of a surface loop with residues 104EW105 forming a b-turn while 106GW107 formed a short anti-parallel b-sheet with the residues 102KN103. 108S is a part of a random coil structure and interacts with L49 that is located in a coiled segment formed by residues 42–50
Summary
Plasmodium falciparum enolase (Pfeno) is a dimeric enzyme with multiple moonlighting functions. Partial restoration of the inter-subunit interactions led to reformation of dimers and restored a significant fraction of the lost enzyme activity These results suggested that the perturbations in the conformation of the surface loop containing the insert sequence were relayed to the interface region, causing dimer dissociation that, in turn, disrupted the enzyme’s active site. Parasite enolase has been shown to act as a cell surface receptor for plasminogen and ligand for mosquito midgut epithelial receptor [14]; a target for biliverdin [15]; and a receptor for peritrophic matrix proteins [16,17] Some of these biochemical activities are likely to be essential for host cell invasion as anti-enolase antibodies neutralize the growth of the parasite in red blood cell stages, conferring partial protection against malaria [18,19]. Antienolase antibodies blocked ookinete traversal through the insect gut epithelium resulting in the failure of parasite transmission [11,13,14]
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