RRC ID 29143
Author Sumida T, Sueyoshi N, Ito M.
Title Molecular cloning and characterization of a novel glucocerebrosidase of Paenibacillus sp. TS12.
Journal J Biochem
Abstract We report here the molecular cloning and characterization of a glucocerebrosidase [EC] from Paenibacillus sp. TS12. The open reading frame of the glucocerebrosidase gene consisted of 2,493 bp nucleotides and encoded 831 amino acid residues. The enzyme exhibited no sequence similarity with a classical glucocerebrosidase belonging to glycoside hydrolase (GH) family 30, but rather showed significant similarity with GH family 3 beta-glucosidases from Clostridium thermocellum, Ruminococcus albus, and Aspergillus aculeateus. The recombinant enzyme, expressed in Escherichia coli BL21(DE3)pLysS, had a molecular weight of 90.7 kDa and hydrolyzed NBD-labeled glucosylceramide, but not galactosylceramide, GM1a or sphingomyelin. The enzyme was most active at pH 6.5, and its apparent Km and Vmax values for NBD-labeled glucosylceramide and p-nitrophenyl-beta-glucopyranoside were 223 microM and 1.60 micromol/min/mg of protein, and 593 microM and 112 micromol/min/mg of protein, respectively. Site-directed mutagenesis indicated that Asp-223 is an essential amino acid for the catalytic reaction and possibly functions a catalytic nucleophile, as in GH family 3 beta-glucosidases. This is the first report of the molecular cloning and characterization of a glucocerebrosidase from a procaryote.
Volume 132(2)
Pages 237-43
Published 2002-8-1
DOI 10.1093/oxfordjournals.jbchem.a003216
PMID 12153721
MeSH Amino Acid Sequence Animals Bacteria / enzymology* Bacteria / genetics Base Sequence Cloning, Molecular Glucosylceramidase / chemistry Glucosylceramidase / genetics Glucosylceramidase / metabolism* Glucosylceramides / metabolism Humans Molecular Sequence Data Molecular Weight Mutation Open Reading Frames Oxadiazoles / metabolism Recombinant Proteins / genetics Recombinant Proteins / metabolism Sequence Alignment Substrate Specificity
IF 2.476
Times Cited 12
DNA material Glc4 (RDB03002) pETGlc4 (RDB03003)