Featured Article - December 2012
Short description: Substrate specificity in trypanosomal sphingolipid synthases can be determined by variation in as few as three residues.
Sphingolipids are essential components of eukaryotic cell membranes and have key roles in protein sorting and signaling, in secondary metabolism, and as precursors in lipid biosynthesis. Sphingolipidoses such as Gaucher and Tay-Sachs diseases are lipid storage disorders that are often fatal during infancy. Furthermore, as protozoan parasites rely on lipid-anchored molecules for protection and infectivity, the interplay with host lipid biosynthesis and metabolism may present an avenue for drug discovery.
Goren, Bangs and Fox (PSI TMPC) have investigated four sphingolipid synthases (TbSLSs) from Trypanosoma brucei, the causative agent of sleeping sickness. TbSLSs are transmembrane enzymes that transfer a phosphoryl headgroup from phospholipid substrates to ceramide to generate sphingolipids. TbSLSs are predicted to contain six transmembrane helices with an arrangement of histidine and aspartic acid residues in loops 3 and 5 composing a HHD motif proposed to be the catalytic triad. Since this set of four TbSLS paralogs produces sphingolipids with the full range of headgroup structures, while also maintaining >90% sequence identity, the observed specificity may be due to a limited number of residues.
As with many membrane proteins, TbSLSs present challenges in generating functional samples. The authors employed their recently developed cell-free translation methods to successfully express and incorporate all four TbSLSs into proteoliposomes for thin-layer chromatography activity assays.
Point mutants of the three HHD residues in TbSLS1 and TbSLS4 confirmed that each of these residues is essential. Noting that the majority of residue differences are adjacent to the HHD triad, the authors examined additional mutants to delineate residues that are responsible for discriminating between the charge and size of different headgroups. Alignment of non-conserved residues in loops 3 and 5 highlighted two candidates. Asp172 in loop 3 is the only negatively charged variant, while the only loop 5 variant, at position 252, is present as either a Phe or Ser. Reciprocal V172D S252F and D172V F252S double mutants of TbSLS1 and TbSLS3, respectively, converted TbSLS1's specificity from anionic to zwitterionic headgroups and likewise exchanged TbSLS3's specificity from zwitterionic to anionic. Probing for determinants of zwitterionic headgroup size selectivity further revealed that the N170A mutation allowed TbSLS2 to accept a smaller headgroup while the A170N N187D double mutation reciprocally allowed TbSLS4 to accept a larger headgroup.
Overall, the experiments reveal that specificity in TbSLS enzymes can be achieved by a remarkably small number of residues and allowed the authors to propose a catalytic mechanism for TbSLS3 and predict the specificity of other trypanosomal SLS enzymes.
M.A. Goren, B.G. Fox, J.D. Bangs. Amino acid determinants of substrate selectivity in the Trypanosoma brucei sphingolipid synthase family.
Biochemistry. 50, 8853-8861 (2011). doi:10.1021/bi200981a