Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site-engineering of sphingomyelin synthases

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dc.contributor.authorKol, Matthijs
dc.contributor.authorPanatala, Radhakrishnan
dc.contributor.authorNordmann, Mirjana
dc.contributor.authorSwart, Leoni
dc.contributor.authorvan Suijlekom, Leonie
dc.contributor.authorCabukusta, Birol
dc.contributor.authorHilderink, Angelika
dc.contributor.authorGrabietz, Tanja
dc.contributor.authorMina, John G. M.
dc.contributor.authorSomerharju, Pentti
dc.contributor.authorKorneev, Sergei
dc.contributor.authorTafesse, Fikadu G.
dc.contributor.authorHolthuis, Joost C. M.
dc.date.accessioned2021-12-23T16:20:07Z-
dc.date.available2021-12-23T16:20:07Z-
dc.date.issued2017
dc.identifier.issn00222275
dc.identifier.urihttps://osnascholar.ub.uni-osnabrueck.de/handle/unios/13333-
dc.description.abstractSM 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.(jlr) 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.
dc.description.sponsorshipEuropean UnionEuropean Commission [299063, 289278]; Deutsche ForschungsgemeinschaftGerman Research Foundation (DFG) [SFB944-P14]; Faculty of Biology/Chemistry from the University of Osnabruck; This work was supported by European Union Seventh Framework Programme Grants 299063 (to J.G.M.M.) and 289278, Deutsche Forschungsgemeinschaft Grant SFB944-P14 (to J.C.M.H.), and an Incentive Award of the Faculty of Biology/Chemistry from the University of Osnabruck (to M.K.).
dc.language.isoen
dc.publisherAMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
dc.relation.ispartofJOURNAL OF LIPID RESEARCH
dc.subjectBiochemistry & Molecular Biology
dc.subjectcell-free expression
dc.subjectCELLS
dc.subjectceramide phosphoethanolamine
dc.subjectCERAMIDE PHOSPHOETHANOLAMINE BIOSYNTHESIS
dc.subjectCHOLESTEROL
dc.subjectclick chemistry
dc.subjectenzyme mechanisms
dc.subjectFAMILY
dc.subjectFUNCTIONAL-CHARACTERIZATION
dc.subjectGolgi apparatus
dc.subjectlipid biochemistry
dc.subjectlipidomics
dc.subjectMICE
dc.subjectmodel membranes
dc.subjectPLASMA-MEMBRANE SPHINGOMYELIN
dc.subjectprotein engineering
dc.subjectPROTEIN SMSR
dc.subjectPURIFICATION
dc.subjectTRANSMEMBRANE DOMAINS
dc.titleSwitching head group selectivity in mammalian sphingolipid biosynthesis by active-site-engineering of sphingomyelin synthases
dc.typejournal article
dc.identifier.doi10.1194/jlr.M076133
dc.identifier.isiISI:000400479800016
dc.description.volume58
dc.description.issue5
dc.description.startpage962
dc.description.endpage973
dc.contributor.orcid0000-0003-0429-1370
dc.contributor.orcid0000-0003-3663-8236
dc.contributor.orcid0000-0002-8575-4164
dc.contributor.orcid0000-0001-8912-1586
dc.contributor.researcheridAAG-2675-2021
dc.contributor.researcheridAAL-9157-2020
dc.contributor.researcheridN-8622-2014
dc.contributor.researcheridAAH-7284-2021
dc.identifier.eissn15397262
dc.publisher.place9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3996 USA
dcterms.isPartOf.abbreviationJ. Lipid Res.
dcterms.oaStatusGreen Accepted, Green Published, hybrid, Green Submitted
crisitem.author.deptFB 05 - Biologie/Chemie-
crisitem.author.deptidfb05-
crisitem.author.parentorgUniversität Osnabrück-
crisitem.author.netidKoSe681-
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