Cloning and expression of gene encoding a novel endoglycoceramidase of Rhodococcus sp. strain C9.
Endoglycoceramidase (EGCase) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides of various glycosphingolipids. We previously reported that the Asn-Glu-Pro (NEP) sequence is part of the active site of EGCase of Rhodococcus sp. strain M-777. This paper describes the molecular cloning of a new EGCase gene utilizing the NEP sequence from the genomic library of Rhodococcus sp. strain C9, which was clearly distinguishable from M-777 by 16S rDNA analysis. C9 EGCase possessed an open reading frame of 1,446 bp encoding 482 amino acids, and showed 78% and 76% identity to M-777 EGCase II at the nucleotide and amino acid levels, respectively. Interestingly, C9 EGCase showed the different specificity to the M-777 enzyme: it hydrolyzed b-series gangliotetraosylceramides more slowly than the M-777 enzyme, whereas both enzymes hydrolyzed a-series gangliosides and neutral glycosphingolipids to the same extent.
Sakaguchi
K
K
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, School Kyushu University, 6-10-1, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Okino
N
N
Sueyoshi
N
N
Izu
H
H
Ito
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D005732:Gangliosides / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D005732:Gangliosides; D006026:Glycoside Hydrolases; C050427:endoglycoceramidase