title: Amino acid determinants of substrate selectivity in the Trypanosoma brucei sphingolipid synthase family.
abstract: The substrate selectivity of four Trypanosoma brucei sphingolipid synthases was examined. TbSLS1, an inositol phosphorylceramide (IPC) synthase, and TbSLS4, a bifunctional sphingomyelin (SM)/ethanolamine phosphorylceramide (EPC) synthase, were inactivated by Ala substitutions of a conserved triad of residues His210, His253, and Asp257 thought to form part of the active site. TbSLS4 also catalyzed the reverse reaction, production of ceramide from sphingomyelin, but none of the Ala substitutions of the catalytic triad in TbSLS4 were able to do so. Site-directed mutagenesis identified residues proximal to the conserved triad that were responsible for the discrimination between charge and size of the different head groups. For discrimination between anionic (phosphoinositol) and zwitterionic (phosphocholine, phosphoethanolamine) head groups, doubly mutated V172D/S252F TbSLS1 and D172V/F252S TbSLS3 showed reciprocal conversion between IPC and bifunctional SM/EPC synthases. For differentiation of zwitterionic headgroup size, N170A TbSLS1 and A170N/N187D TbSLS4 showed reciprocal conversion between EPC and bifunctional SM/EPC synthases. These studies provide a mapping of the SLS active site and demonstrate that differences in catalytic specificity of the T. brucei enzyme family are controlled by natural variations in as few as three residue positions.
authors: Department of Biochemistry,School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors: Michael A
authors: MA
authors: Goren
authors: Brian G
authors: BG
authors: Fox
authors: James D
authors: JD
authors: Bangs
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D002384:Catalysis; D020134:Catalytic Domain; D004983:Ethanolamines / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D008956:Models, Chemical; D009097:Multienzyme Complexes / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016366:Open Reading Frames; D010767:Phosphorylcholine / Q000737:chemistry; D010770:Phosphotransferases / Q000737:chemistry; D013109:Sphingomyelins / Q000737:chemistry; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry*; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
chemical_list: D000596:Amino Acids; D004983:Ethanolamines; D008081:Liposomes; D009097:Multienzyme Complexes; D013109:Sphingomyelins; D010767:Phosphorylcholine; C005448:phosphorylethanolamine; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C070186:phosphatidylcholine-ceramide phosphocholine transferase