The Glu residue in the conserved Asn-Glu-Pro sequence of endoglycoceramidase is essential for enzymatic activity.
Endoglycoceramidase (EGCase) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides of various glycosphingolipids. We previously cloned the gene encoding EGCase II of Rhodococcus sp. M-777 and reported that the deduced amino acid sequence contained the Asn-Glu-Pro (NEP) sequence, conserved as part of the active site of family A cellulases (endo-1,4-beta-glucanases) (J. Biol. Chem. 272, 19846, 1997). The NEP sequence was also found in the deduced amino acid sequence of the newly cloned EGCase gene of Rhodococcus sp. C9. Replacement of the Glu residue in the NEP sequence with Gln or Asp by site-directed mutagenesis caused marked loss of enzymatic activity in both the M-777 and C9 EGCases but did not affect the expression of EGCase protein. This result clearly indicated that the NEP sequence is part of the active site of EGCase, in which the Glu residue plays an important role in the catalytic reaction, possibly in the same manner as in endo-1,4-beta-glucanase.
Sakaguchi
K
K
Department of Bioscience and Biotechnology, Division of Bioresource and Bioenvironmental Sciences, Graduate School Kyushu University, 6-10-1, Hakozaki, Higashi-ku, Fukuoka, 812-8581, Japan.
Okino
N
N
Izu
H
H
Ito
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D002240:Carbohydrate Sequence; D005456:Fluorescent Dyes / Q000494:pharmacology; D005973:Glutamine / Q000502:physiology*; D006026:Glycoside Hydrolases / Q000737:chemistry*; D006868:Hydrolysis; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010069:Oxadiazoles / Q000494:pharmacology; D012240:Rhodococcus / Q000201:enzymology; D013997:Time Factors
D000596:Amino Acids; D005456:Fluorescent Dyes; D010069:Oxadiazoles; D005973:Glutamine; C011514:4-nitrobenzofurazan; D006026:Glycoside Hydrolases; C050427:endoglycoceramidase