Mechanistic insights into the hydrolysis and synthesis of ceramide by neutral ceramidase

Tsuyoshi Inoue, Nozomu Okino, Yoshimitsu Kakuta, Atsushi Hijikata, Hiroyuki Okano, Hatsumi M. Goda, Motohiro Tani, Noriyuki Sueyoshi, Kouji Kambayashi, Hiroyoshi Matsumura, Yasushi Kai, Makoto Ito

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22 Citations (Scopus)

Abstract

Ceramidase (CDase; EC 3.5.1.23) hydrolyzes ceramide to generate sphingosine and fatty acid. The enzyme plays a regulatory role in a variety of physiological events in eukaryotes and also functions as an exotoxin in particular bacteria. The crystal structures of neutral CDase from Pseudomonas aeruginosa (PaCD) in the C2-ceramide-bound and -unbound forms were determined at 2.2 and 1.4 Å resolutions, respectively. PaCD consists of two domains, and the Zn2+- and Mg2+/Ca2+-binding sites are found within the center of the N-terminal domain and the interface between the domains, respectively. The structural comparison between the C2- ceramide-bound and unbound forms revealed an open-closed conformational change occurring to loop I upon binding of C2-ceramide. In the closed state, this loop sits above the Zn2+ coordination site and over the opening to the substrate binding site. Mutational analyses of residues surrounding the Zn2+ of PaCD and rat neutral CDase revealed that the cleavage or creation of the N-acyl linkage of ceramide follows a similar mechanism as observed for the Zn2+-dependent carboxypeptidases. The results provide an understanding of the molecular mechanism of hydrolysis and synthesis of ceramide by the enzyme. Furthermore, insights into the actions of PaCD and eukaryotic neutral CDases as an exotoxin and mediators of sphingolipid signaling are also revealed, respectively.

Original languageEnglish
Pages (from-to)9566-9577
Number of pages12
JournalJournal of Biological Chemistry
Volume284
Issue number14
DOIs
Publication statusPublished - Apr 3 2009

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Inoue, T., Okino, N., Kakuta, Y., Hijikata, A., Okano, H., Goda, H. M., Tani, M., Sueyoshi, N., Kambayashi, K., Matsumura, H., Kai, Y., & Ito, M. (2009). Mechanistic insights into the hydrolysis and synthesis of ceramide by neutral ceramidase. Journal of Biological Chemistry, 284(14), 9566-9577. https://doi.org/10.1074/jbc.M808232200