A novel endoglycoceramidase hydrolyzes oligogalactosylceramides to produce galactooligosaccharides and ceramides.
Enzymes capable of hydrolyzing the beta-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 (beta/alpha)8 barrel structure in which Glu234 at the end of beta-strand 4 and Glu341 at the end of beta-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 beta-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 lactoseries glycosphingolipids. The enzyme is therefore systematically named "oligogalactosyl-N-acylsphingosine 1,1'-beta-galactohydrolase" or tentatively designated "endogalactosylceramidase."
Ishibashi
Yohei
Y
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Nakasone
Toru
T
Kiyohara
Masashi
M
Horibata
Yasuhiro
Y
Sakaguchi
Keishi
K
Hijikata
Atsushi
A
Ichinose
Sachiyo
S
Omori
Akira
A
Yasui
Yasuyuki
Y
Imamura
Akihiro
A
Ishida
Hideharu
H
Kiso
Makoto
M
Okino
Nozomu
N
Ito
Makoto
M
D000595:Amino Acid Sequence; D002384:Catalysis; D002518:Ceramides / Q000378:metabolism*; D003001:Cloning, Molecular; D005690:Galactose / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016929:Rhodococcus equi / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D002518:Ceramides; D009844:Oligosaccharides; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C050427:endoglycoceramidase; D005690:Galactose