Mechanistic insights into the hydrolysis and synthesis of ceramide by neutral ceramidase.
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 A resolutions, respectively. PaCD consists of two domains, and the Zn(2+)- and Mg(2+)/Ca(2+)-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 Zn(2+) coordination site and over the opening to the substrate binding site. Mutational analyses of residues surrounding the Zn(2+) 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 Zn(2+)-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.
Inoue
Tsuyoshi
T
Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-Oka, Suita, Osaka 565-0871, Japan. inouet@chem.eng.osaka-u.ac.jp
Okino
Nozomu
N
Kakuta
Yoshimitsu
Y
Hijikata
Atsushi
A
Okano
Hiroyuki
H
Goda
Hatsumi M
HM
Tani
Motohiro
M
Sueyoshi
Noriyuki
N
Kambayashi
Kouji
K
Matsumura
Hiroyoshi
H
Kai
Yasushi
Y
Ito
Makoto
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D002460:Cell Line; D002518:Ceramides / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D006801:Humans; D006868:Hydrolysis; D008670:Metals / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D055576:Neutral Ceramidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D002518:Ceramides; D008670:Metals; D055576:Neutral Ceramidase