Molecular cloning and characterization of a novel glucocerebrosidase of Paenibacillus sp. TS12.
We report here the molecular cloning and characterization of a glucocerebrosidase [EC 3.2.1.45] 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.
Sumida
Tomomi
T
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Sueyoshi
Noriyuki
N
Ito
Makoto
M
D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D005962:Glucosylceramidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005963:Glucosylceramides / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D016366:Open Reading Frames; D010069:Oxadiazoles / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
D005963:Glucosylceramides; D010069:Oxadiazoles; D011994:Recombinant Proteins; C011514:4-nitrobenzofurazan; D005962:Glucosylceramidase