title: Characterization of homologous sphingosine-1-phosphate lyase isoforms in the bacterial pathogen Burkholderia pseudomallei.
abstract: Sphingolipids (SLs) are ubiquitous elements in eukaryotic membranes and are also found in some bacterial and viral species. As well as playing an integral structural role, SLs also act as potent signaling molecules involved in numerous cellular pathways and have been linked to many human diseases. A central SL signaling molecule is sphingosine-1-phosphate (S1P), whose breakdown is catalyzed by S1P lyase (S1PL), a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the cleavage of S1P to (2E)-hexadecenal (2E-HEX) and phosphoethanolamine. Here, we show that the pathogenic bacterium, Burkholderia pseudomallei K96243, encodes two homologous proteins (S1PL2021 and S1PL2025) that display moderate sequence identity to known eukaryotic and prokaryotic S1PLs. Using an established MS-based methodology, we show that recombinant S1PL2021 is catalytically active. We also used recombinant human fatty aldehyde dehydrogenase to develop a spectrophotometric enzyme-coupled assay to detect 2E-HEX formation and measure the kinetic constants of the two B. pseudomallei S1PL isoforms. Furthermore, we determined the X-ray crystal structure of the PLP-bound form of S1PL2021 at 2.1 Å resolution revealing that the enzyme displays a conserved structural fold and active site architecture comparable with known S1PLs. The combined data suggest that B. pseudomallei has the potential to degrade host SLs in a S1PL-dependent manner.
authors: EastChem School of Chemistry University of Edinburgh, Edinburgh EH9 3FJ, United Kingdom.
authors: Christopher J
authors: CJ
authors: McLean
authors: Institute of Quantitative Biology, Biochemistry, and Biotechnology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3FJ, United Kingdom.
authors: Jon
authors: J
authors: Marles-Wright
authors: School of Biosciences, University of Exeter, Exeter EX4 4QD, United Kingdom.
authors: Rafael
authors: R
authors: Custodio
authors: EastChem School of Chemistry University of Edinburgh, Edinburgh EH9 3FJ, United Kingdom.
authors: Jonathan
authors: J
authors: Lowther
authors: EastChem School of Chemistry University of Edinburgh, Edinburgh EH9 3FJ, United Kingdom.
authors: Amanda J
authors: AJ
authors: Kennedy
authors: EastChem School of Chemistry University of Edinburgh, Edinburgh EH9 3FJ, United Kingdom.
authors: Jacob
authors: J
authors: Pollock
authors: EastChem School of Chemistry University of Edinburgh, Edinburgh EH9 3FJ, United Kingdom.
authors: David J
authors: DJ
authors: Clarke
authors: School of Biosciences, University of Exeter, Exeter EX4 4QD, United Kingdom.
authors: Alan R
authors: AR
authors: Brown
authors: EastChem School of Chemistry University of Edinburgh, Edinburgh EH9 3FJ, United Kingdom Dominic.Campopiano@ed.ac.uk.
authors: Dominic J
authors: DJ
authors: Campopiano
mesh_terms: D000446:Aldehyde-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016957:Burkholderia pseudomallei / Q000737:chemistry / Q000201:enzymology*; D018360:Crystallography, X-Ray; D008246:Lysophospholipids / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics*; D011732:Pyridoxal Phosphate / Q000737:chemistry; D013107:Sphingolipids / Q000737:chemistry / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism
chemical_list: D008246:Lysophospholipids; D020033:Protein Isoforms; D013107:Sphingolipids; C060506:sphingosine 1-phosphate; D011732:Pyridoxal Phosphate; D000446:Aldehyde-Lyases; C022649:sphingosine 1-phosphate lyase (aldolase); D013110:Sphingosine
keywords: assay; fatty aldehyde dehydrogenase; pyridoxal 5′-phosphate; sphingolipids