Sequence and expression of Thai rosewood beta-glucosidase/beta-fucosidase,a family 1 glycosyl hydrolase glycoprotein

Dalcochinin-8'-O-beta -glucoside beta -glucosidase (dalcochinase) from the Thai rosewood (Dalbergia cochinchinensis Pierre) has aglycone specificity f or isoflavonoids and can hydrolyze both P-glucosides and beta -fucosides. T o determine its structure and evolutionary lineage, the sequence of the enz yme was determined by peptide sequencing followed by PCR cloning, The cDNA included a reading frame coding for 547 amino acids including a 23 amino ac id 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% ident ical to the cyanogenic p-glucosidase from white clover glycosyl hydrolase f amily 1, for which an Xray crystal structure has been solved. Based on this homology, residues which may contribute to the different substrate specifi cities of the two enzymes were identified. Eight putative glycosylation sit es were identified, and one was confirmed to be glycosylated by Edman degra dation and mass spectrometry, The protein was expressed as a prepro-alpha - mating factor fusion in Pichia pastoris, and the activity of the secreted e nzyme was characterized. The recombinant enzyme and the enzyme purified fro m seeds showed the same K-m for pNP-glucoside and pNP-fucoside, had the sam e ratio of V-max for these substrates, and similarly hydrolyzed the natural substrate, dalcochinin-8'-beta -glucoside.