title: Localization and nucleotide specificity of Blastocystis succinyl-CoA synthetase.
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.
authors.affiliation: School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK.
authors.forename: Karleigh
authors.initials: K
authors.lastname: Hamblin
authors.forename: Daron M
authors.initials: DM
authors.lastname: Standley
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Rogers
authors.forename: Alexandra
authors.initials: A
authors.lastname: Stechmann
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Roger
authors.forename: Robin
authors.initials: R
authors.lastname: Maytum
authors.forename: Mark
authors.initials: M
authors.lastname: van der Giezen
mesh_terms: D000818:Animals; D001483:Base Sequence; D016844:Blastocystis / Q000737:chemistry / Q000166:cytology* / Q000201:enzymology* / Q000235:genetics; D016776:Blastocystis Infections / Q000469:parasitology; D022083:Cytoplasmic Structures / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011685:Purine Nucleotides / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity; D013389:Succinate-CoA Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013552:Swine / Q000235:genetics
chemical_list: D021122:Protein Subunits; D015800:Protozoan Proteins; D011685:Purine Nucleotides; D013389:Succinate-CoA Ligases