Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site-engineering of sphingomyelin synthases.
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, SMS-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate the 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 Glu permitting SMS-catalyzed CPE production and Asp 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.
Molecular Cell Biology Division, Department of Biology/Chemistry, University of Osnabrück, 49076 Osnabrück, Germany matthijs.kol@biologie.uni-osnabrueck.de holthuis@uos.de.|Membrane Biochemistry and Biophysics, Bijvoet Center and Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands.
Matthijs
M
Kol
Molecular Cell Biology Division, Department of Biology/Chemistry, University of Osnabrück, 49076 Osnabrück, Germany.|Membrane Biochemistry and Biophysics, Bijvoet Center and Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands.
Radhakrishnan
R
Panatala
Molecular Cell Biology Division, Department of Biology/Chemistry, University of Osnabrück, 49076 Osnabrück, Germany.
Mirjana
M
Nordmann
Membrane Biochemistry and Biophysics, Bijvoet Center and Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands.
Leoni
L
Swart
Membrane Biochemistry and Biophysics, Bijvoet Center and Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands.
Leonie
L
van Suijlekom
Molecular Cell Biology Division, Department of Biology/Chemistry, University of Osnabrück, 49076 Osnabrück, Germany.
Birol
B
Cabukusta
Molecular Cell Biology Division, Department of Biology/Chemistry, University of Osnabrück, 49076 Osnabrück, Germany.
Angelika
A
Hilderink
Molecular Cell Biology Division, Department of Biology/Chemistry, University of Osnabrück, 49076 Osnabrück, Germany.
Tanja
T
Grabietz
Molecular Cell Biology Division, Department of Biology/Chemistry, University of Osnabrück, 49076 Osnabrück, Germany.
John G M
JGM
Mina
Medical Biochemistry, Institute of Biomedicine, University of Helsinki, Helsinki 00014, Finland.
Pentti
P
Somerharju
Molecular Cell Biology Division, Department of Biology/Chemistry, University of Osnabrück, 49076 Osnabrück, Germany.
Sergei
S
Korneev
Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR 97239.
Fikadu G
FG
Tafesse
Molecular Cell Biology Division, Department of Biology/Chemistry, University of Osnabrück, 49076 Osnabrück, Germany matthijs.kol@biologie.uni-osnabrueck.de holthuis@uos.de.|Membrane Biochemistry and Biophysics, Bijvoet Center and Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands.
Joost C M
JCM
Holthuis
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D006801:Humans; D016297:Mutagenesis, Site-Directed; D000072417:Protein Domains; D013107:Sphingolipids / Q000378:metabolism*; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D013107:Sphingolipids; D017855:Transferases (Other Substituted Phosphate Groups); C070186:phosphatidylcholine-ceramide phosphocholine transferase
Golgi apparatus; cell-free expression; ceramide phosphoethanolamine; click chemistry; enzyme mechanisms; lipid biochemistry; lipidomics; model membranes; protein engineering