TY - JOUR
T1 - A novel endoglycoceramidase hydrolyzes oligogalactosylceramides to produce galactooligosaccharides and ceramides
AU - Ishibashi, Yohei
AU - Nakasone, Toru
AU - Kiyohara, Masashi
AU - Horibata, Yasuhiro
AU - Sakaguchi, Keishi
AU - Hijikata, Atsushi
AU - Ichinose, Sachiyo
AU - Omori, Akira
AU - Yasui, Yasuyuki
AU - Imamura, Akihiro
AU - Ishida, Hideharu
AU - Kiso, Makoto
AU - Okino, Nozomu
AU - Ito, Makoto
PY - 2007/4/13
Y1 - 2007/4/13
N2 - Enzymes capable of hydrolyzing the β-glycosidic linkage between oligosaccharides and ceramides in various glycosphingolipids has been found in microorganisms and invertebrates and designated endoglycoceramidase (EC 3.2.1.123) or ceramide glycanase. Here we report the molecular cloning, characterization, and homology modeling of a novel endoglycoceramidase that hydrolyzes oligogalactosylceramides to produce galactooligosaccharides and ceramides. The novel enzyme was purified from a culture supernatant of Rhodococcus equi, and the gene encoding 488 deduced amino acids was cloned using peptide sequences of the purified enzyme. Eight residues essential for the catalytic reaction in microbial and animal endoglycoceramidases were all conserved in the deduced amino acid sequence of the novel enzyme. Homology modeling of the enzyme using endocellulase E1 as a template revealed that the enzyme displays a (β/α)8 barrel structure in which Glu234 at the end of β-strand 4 and Glu341 at the end of β-strand 7 could function as an acid/base catalyst and a nucleophile, respectively. Site-directed mutagenesis of these glutamates resulted in a complete loss of the activity without a change in their CD spectra. The recombinant enzyme hydrolyzed the β-galactosidic linkage between oligosaccharides and ceramides of 6-gala series glycosphingolipids that were completely resistant to hydrolysis by the enzymes reported so far. In contrast, the novel enzyme did not hydrolyze ganglio-, globo-, or lacto- series glycosphingolipids. The enzyme is therefore systematically named "oligogalactosyl-N-acylsphingosine 1,1′-β-galactohydrolase" or tentatively designated "endogalactosylceramidase."
AB - Enzymes capable of hydrolyzing the β-glycosidic linkage between oligosaccharides and ceramides in various glycosphingolipids has been found in microorganisms and invertebrates and designated endoglycoceramidase (EC 3.2.1.123) or ceramide glycanase. Here we report the molecular cloning, characterization, and homology modeling of a novel endoglycoceramidase that hydrolyzes oligogalactosylceramides to produce galactooligosaccharides and ceramides. The novel enzyme was purified from a culture supernatant of Rhodococcus equi, and the gene encoding 488 deduced amino acids was cloned using peptide sequences of the purified enzyme. Eight residues essential for the catalytic reaction in microbial and animal endoglycoceramidases were all conserved in the deduced amino acid sequence of the novel enzyme. Homology modeling of the enzyme using endocellulase E1 as a template revealed that the enzyme displays a (β/α)8 barrel structure in which Glu234 at the end of β-strand 4 and Glu341 at the end of β-strand 7 could function as an acid/base catalyst and a nucleophile, respectively. Site-directed mutagenesis of these glutamates resulted in a complete loss of the activity without a change in their CD spectra. The recombinant enzyme hydrolyzed the β-galactosidic linkage between oligosaccharides and ceramides of 6-gala series glycosphingolipids that were completely resistant to hydrolysis by the enzymes reported so far. In contrast, the novel enzyme did not hydrolyze ganglio-, globo-, or lacto- series glycosphingolipids. The enzyme is therefore systematically named "oligogalactosyl-N-acylsphingosine 1,1′-β-galactohydrolase" or tentatively designated "endogalactosylceramidase."
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U2 - 10.1074/jbc.M608445200
DO - 10.1074/jbc.M608445200
M3 - Article
C2 - 17244618
AN - SCOPUS:34249723698
VL - 282
SP - 11386
EP - 11396
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 15
ER -