Abstract
The anaerobic lifestyle of the intestinal parasite Blastocystis raises questions about the biochemistry and function of its mitochondria-like organelles. We have characterized the Blastocystis succinyl-CoA synthetase (SCS), a tricarboxylic acid cycle enzyme that conserves energy by substrate-level phosphorylation. We show that SCS localizes to the enigmatic Blastocystis organelles, indicating that these organelles might play a similar role in energy metabolism as classic mitochondria. Although analysis of residues inside the nucleotide-binding site suggests that Blastocystis SCS is GTP-specific, we demonstrate that it is ATP-specific. Homology modelling, followed by flexible docking and molecular dynamics simulations, indicates that while both ATP and GTP fit into the Blastocystis SCS active site, GTP is destabilized by electrostatic dipole interactions with Lys 42 and Lys 110, the side-chains of which lie outside the nucleotide-binding cavity. It has been proposed that residues in direct contact with the substrate determine nucleotide specificity in SCS. However, our results indicate that, in Blastocystis, an electrostatic gatekeeper controls which ligands can enter the binding site.
Highlights
Blastocystis is a widespread human intestinal parasite infecting up to 10% of the developed world (Stenzel and Boreham, 1996)
Homology modelling, followed by flexible docking and molecular dynamics simulations, indicates that while both ATP and GTP fit into the Blastocystis succinyl-CoA synthetase (SCS) active site, GTP is destabilized by electrostatic dipole interactions with Lys 42 and Lys 110, the side-chains of which lie outside the nucleotide-binding cavity
We have broadened the taxonomic breadth of SCS research and characterized the first stramenopile SCS from the human intestinal parasite Blastocystis
Summary
Blastocystis is a widespread human intestinal parasite infecting up to 10% of the developed world (Stenzel and Boreham, 1996). The role Blastocystis has in causing actual disease, if any, is still a matter of dispute. It is one of the few known human parasites within the stramenopiles (Silberman et al, 1996), a group containing, for example, diatoms, oomycetes and brown algae such as the Californian giant kelp. Blastocystis is a strict anaerobe (Zierdt, 1991), it has been shown to contain organelles that have features traditionally associated with mitochondria, such as cristae, a membrane potential and an associated organellar genome (Zierdt, 1991; Nasirudeen and Tan, 2004). These organelles have features normally associated with anaerobic mitochondria-related organelles, such as hydrogenosomes and mitosomes (see van der Giezen et al, 2005 for a review), with the apparent absence of several tricarboxylic acid (TCA) cycle enzymes and cytochromes (Zierdt, 1986)
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