TY - JOUR
T1 - The Glu residue in the conserved Asn-Glu-Pro sequence of endoglycoceramidase is essential for enzymatic activity
AU - Sakaguchi, Keishi
AU - Okino, Nozomu
AU - Izu, Hiroyuki
AU - Ito, Makoto
N1 - Funding Information:
We thank Dr. Takashi Nakamura of Kyushu University and Dr. Tatsuya Yamagata of Japan Instate of Leather Research for their encouragement throughout the course of this study. This work was supported in part by Grants-in-aid for Scientific Research 09460051 from the Ministry of Education, Science and Culture of Japan.
PY - 1999/6/24
Y1 - 1999/6/24
N2 - 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-β-glucanases). 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-β-glucanase.
AB - 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-β-glucanases). 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-β-glucanase.
UR - https://www.scopus.com/pages/publications/0033600140
UR - https://www.scopus.com/pages/publications/0033600140#tab=citedBy
U2 - 10.1006/bbrc.1999.0855
DO - 10.1006/bbrc.1999.0855
M3 - Article
C2 - 10381348
AN - SCOPUS:0033600140
SN - 0006-291X
VL - 260
SP - 89
EP - 93
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 1
ER -