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 acc...

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Bibliographic Details
Published in:Biochimie 2008-10, Vol.90 (10), p.1560-1565
Main Authors: Lidome, Emilie, Graf, Christine, Jaritz, Markus, Schanzer, Andrea, Rovina, Philipp, Nikolay, Rainer, Bornancin, Frédéric
Format: Article
Language:English
Subjects:
Amino Acid Motifs
Amino Acid Sequence
Animals
Catalytic Domain
Ceramide metabolism
Cercopithecus aethiops
Conserved Sequence
COS Cells
Cysteine - metabolism
Cysteine motif
Enzyme activity
Humans
Molecular Sequence Data
Oxidation
Oxidation-Reduction
Phosphotransferases (Alcohol Group Acceptor) - chemistry
Phosphotransferases (Alcohol Group Acceptor) - metabolism
Sulfhydryl Compounds
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Summary: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.
ISSN:0300-9084
1638-6183
DOI:10.1016/j.biochi.2008.07.001