We found a match
Your institution may have rights to this item. Sign in to continue.
- Title
Sequence and Expression of Thai Rosewood β-Glucosidase/β-Fucosidase, a Family 1 Glycosyl Hydrolase Glycoprotein1.
- Authors
Cairns, James R. Ketudat; Champattanachai, Voraratt; Srisomsap, Chantragan; Wittman-Liebold, Brigitte; Thiede, Bernd; Svasti, Jisnuson
- Abstract
Dalcochinin-8′-O-β-glucoside β-glucosidase (dalcochinase) from the Thai rosewood (Dalbergia cochinchinensis Pierre) has aglycone specificity for isoflavonoids and can hydrolyze both β-glucosides and β-fucosides. To determine its structure and evolutionary lineage, the sequence of the enzyme was determined by peptide sequencing followed by PCR cloning. The cDNA included a reading frame coding for 547 amino acids including a 23 amino acid propeptide and a 524 amino acid mature protein. The sequences determined at peptide level were found in the cDNA sequence, indicating the sequence obtained was indeed the dalcochinase enzyme. The mature enzyme is 60% identical to the cyanogenic β-glucosidase from white clover glycosyl hydrolase family 1, for which an X-ray crystal structure has been solved. Based on this homology, residues which may contribute to the different substrate specificities of the two enzymes were identified. Eight putative glycosylation sites were identified, and one was confirmed to be glycosylated by Edman degradation and mass spectrometry. The protein was expressed as a prepro-α-mating factor fusion in Pichia pastoris, and the activity of the secreted enzyme was characterized. The recombinant enzyme and the enzyme purified from seeds showed the same Em for pNP-glucoside and pNP-fucoside, had the same ratio of Vmax for these substrates, and similarly hydrolyzed the natural substrate, dalcochinin-8′-β-glucoside.
- Subjects
GLUCOSIDASES; ISOFLAVONOIDS; ANTISENSE DNA; AMINO acids; ENZYMES
- Publication
Journal of Biochemistry, 2000, Vol 128, Issue 6, p999
- ISSN
0021-924X
- Publication type
Article
- DOI
10.1093/oxfordjournals.jbchem.a022852