title: Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site engineering of sphingomyelin synthases.
abstract: SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase (SMS) 1 in the Golgi and SMS2 at the plasma membrane. Mammalian cells also synthesize trace amounts of the SM analog ceramide phosphoethanolamine (CPE), but the physiological relevance of CPE production is unclear. Previous work revealed that SMS2 is a bifunctional enzyme producing both SM and CPE, whereas a closely related enzyme, sphingomyelin synthase-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmatic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate head group selectivity of SMS family members. Notably, a single residue adjacent to the catalytic histidine in the third exoplasmic loop profoundly influenced enzyme specificity, with glutamic acid permitting SMS-catalyzed CPE production and aspartic acid confining the enzyme to produce SM. An exchange of exoplasmic residues with SMSr proved sufficient to convert SMS1 into a bulk CPE synthase. This allowed us to establish mammalian cells that produce CPE rather than SM as the principal phosphosphingolipid and provide a model of the molecular interactions that impart catalytic specificity among SMS enzymes.
authors.affiliation: Molecular Cell Biology Division, Department of Biology/Chemistry, University of Osnabrück, 49076 Osnabrück, Germany Membrane Biochemistry & Biophysics, Bijvoet Center and Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands.
authors.forename: Matthijs
authors.initials: M
authors.lastname: Kol
authors.affiliation: Molecular Cell Biology Division, Department of Biology/Chemistry, University of Osnabrück, 49076 Osnabrück, Germany Membrane Biochemistry & Biophysics, Bijvoet Center and Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands.
authors.forename: Radhakrishnan
authors.initials: R
authors.lastname: Panatala
authors.affiliation: Molecular Cell Biology Division, Department of Biology/Chemistry, University of Osnabrück, 49076 Osnabrück, Germany.
authors.forename: Mirjana
authors.initials: M
authors.lastname: Nordmann
authors.affiliation: Membrane Biochemistry & Biophysics, Bijvoet Center and Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands.
authors.forename: Leoni
authors.initials: L
authors.lastname: Swart
authors.affiliation: Membrane Biochemistry & Biophysics, Bijvoet Center and Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands.
authors.forename: Leonie
authors.initials: L
authors.lastname: van Suijlekom
authors.affiliation: Molecular Cell Biology Division, Department of Biology/Chemistry, University of Osnabrück, 49076 Osnabrück, Germany.
authors.forename: Birol
authors.initials: B
authors.lastname: Cabukusta
authors.affiliation: Molecular Cell Biology Division, Department of Biology/Chemistry, University of Osnabrück, 49076 Osnabrück, Germany.
authors.forename: Angelika
authors.initials: A
authors.lastname: Hilderink
authors.affiliation: Molecular Cell Biology Division, Department of Biology/Chemistry, University of Osnabrück, 49076 Osnabrück, Germany.
authors.forename: Tanja
authors.initials: T
authors.lastname: Grabietz
authors.affiliation: Molecular Cell Biology Division, Department of Biology/Chemistry, University of Osnabrück, 49076 Osnabrück, Germany.
authors.forename: John G M
authors.initials: JG
authors.lastname: Mina
authors.affiliation: Medical Biochemistry, Institute of Biomedicine, University of Helsinki, Helsinki 00014, Finland.
authors.forename: Pentti
authors.initials: P
authors.lastname: Somerharju
authors.affiliation: Molecular Cell Biology Division, Department of Biology/Chemistry, University of Osnabrück, 49076 Osnabrück, Germany.
authors.forename: Sergei
authors.initials: S
authors.lastname: Korneev
authors.affiliation: Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, OR 97239.
authors.forename: Fikadu G
authors.initials: FG
authors.lastname: Tafesse
authors.affiliation: Molecular Cell Biology Division, Department of Biology/Chemistry, University of Osnabrück, 49076 Osnabrück, Germany Membrane Biochemistry & Biophysics, Bijvoet Center and Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands holthuis@uos.de.
authors.forename: Joost C M
authors.initials: JC
authors.lastname: Holthuis
mesh_terms: D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002474:Cell-Free System; D057930:Click Chemistry; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006056:Golgi Apparatus / Q000201:enzymology; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics*; D015202:Protein Engineering; D013109:Sphingomyelins / Q000096:biosynthesis* / Q000235:genetics; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry / Q000235:genetics*
chemical_list: D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D013109:Sphingomyelins; C060281:ceramide phosphoethanolamine; C000592293:SAMD8 protein, human; C455440:SGMS1 protein, human; D017855:Transferases (Other Substituted Phosphate Groups)
keywords: Golgi apparatus; cell-free expression; ceramide phosphoethanolamine; click chemistry; enzyme mechanisms; lipid biochemistry; lipidomics; model membranes; protein engineering