Molecular cloning and bacterial expression of a cDNA encoding furostanol glycoside 26-O-beta-glucosidase of Costus speciosus.
Furostanol glycoside 26-O-beta-glucosidase (F26G) purified from Costus speciosus rhizomes was digested with endoproteinase, and several internal peptide fragments were obtained. Degenerate oligonucleotide primers based on amino acid sequences of the peptides were used for amplification of F26G cDNA fragments by applying nested polymerase chain reactions to cDNAs from in vitro cultured plantlets of C. speciosus. Using primers based on sequences of the cDNA fragments, the 5'- and 3'-end clones were isolated by rapid amplification of cDNA ends (RACE) methods. Finally, the entire coding portion of F26G cDNA was cloned by using primers designed from sequences of the RACE products. The deduced amino acid sequence of CSF26G1, the protein encoded by the cloned cDNA, consists of 562 amino acids and shows high homology to a widely distributed family of beta-glucosidases (BGA family). Cell-free homogenate of Escherichia coli expressing CSF26G1 cDNA showed beta-glucosidase activity specific for cleavage of the C-26 glucosidic bond of furostanol glycosides.
Inoue
K
K
Department of Pharmacognosy and Phytochemistry, Faculty of Pharmaceutical Sciences, The University of Tokyo, Japan.
Shibuya
M
M
Yamamoto
K
K
Ebizuka
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D017343:Genes, Plant; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010944:Plants / Q000201:enzymology / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013150:Spirostans / Q000378:metabolism; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013150:Spirostans; C044934:gracillin; C097713:furostanol glycoside 26-O-beta-glucosidase; D001617:beta-Glucosidase; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase