title: A conserved cysteine motif essential for ceramide kinase function.
abstract: Ceramide kinase (CerK) is a sphingolipid metabolizing enzyme very sensitive to oxidation; however, the determinants are unknown. We show here that the thiol-modifying agent N-ethyl-maleimide abrogates CerK activity in vitro and in a cell based assay, implying that important cysteine residues are accessible in purified as well as endogenous CerK. We replaced every 22 residues in human CerK, by an alanine, and measured activity in the resulting mutant proteins. This led to identification of a cluster of cysteines, C(347)XXXC(351)XXC(354), essential for CerK function. These findings are discussed based on homology modeling of the catalytic domain of CerK.
authors.affiliation: Novartis Institutes for BioMedical Research, Vienna, Vienna, Austria.
authors.forename: Emilie
authors.initials: E
authors.lastname: Lidome
authors.forename: Christine
authors.initials: C
authors.lastname: Graf
authors.forename: Markus
authors.initials: M
authors.lastname: Jaritz
authors.forename: Andrea
authors.initials: A
authors.lastname: Schanzer
authors.forename: Philipp
authors.initials: P
authors.lastname: Rovina
authors.forename: Rainer
authors.initials: R
authors.lastname: Nikolay
authors.forename: Frédéric
authors.initials: F
authors.lastname: Bornancin
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D020134:Catalytic Domain; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D003545:Cysteine / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000378:metabolism*; D013438:Sulfhydryl Compounds
chemical_list: D013438:Sulfhydryl Compounds; D017853:Phosphotransferases (Alcohol Group Acceptor); C066324:ceramide kinase; D003545:Cysteine