A conserved cysteine motif essential for ceramide kinase function.
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.
Lidome
Emilie
E
Novartis Institutes for BioMedical Research, Vienna, Vienna, Austria.
Graf
Christine
C
Jaritz
Markus
M
Schanzer
Andrea
A
Rovina
Philipp
P
Nikolay
Rainer
R
Bornancin
Frédéric
F
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
D013438:Sulfhydryl Compounds; D017853:Phosphotransferases (Alcohol Group Acceptor); C066324:ceramide kinase; D003545:Cysteine