Sequence and Expression of Thai Rosewood  -Glucosidase/ -Fucosidase, a Family 1 Glycosyl Hydrolase Glycoprotein

Abstract

Dalcochinin-8'-O-beta-glucoside beta-glucosidase (dalcochinase) from the Thai rosewood (Dalbergia cochinchinensis Pierre) has aglycone specificity for isoflavonoids and can hydrolyze both beta-glucosides and beta-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 beta-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-alpha-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 K(m) for pNP-glucoside and pNP-fucoside, had the same ratio of V(max) for these substrates, and similarly hydrolyzed the natural substrate, dalcochinin-8'-beta-glucoside.

The β-Glucosidases Responsible for Bioactivation of Hydroxynitrile Glucosides in Lotus japonicus

“…This cross-reactivity is consistent with previous observations that almond (Prunus dulcis) b-glucosidase hydrolyzes daidzin (Ismail and Hayes, 2005;Chuankhayan et al, 2007a), whereas b-glucosidases hydrolyzing isoflavonoid glucosides have not been found to hydrolyze cyanogenic glucosides (Ketudat Cairns et al, 2000;Chuankhayan et al, 2005). The isoflavonoid glucoside profile in L. japonicus is currently not known, and daidzin has not been reported to be present Suzuki et al, 2008).…”

Genome mining and motif modifications of glycoside hydrolase family 1 members encoded by Geobacillus kaustophilus HTA426 provide thermostable 6-phospho-β-glycosidase and β-fucosidase

“…β-Fucosidase activity was observed over a wide pH range (4.0-9.0) with an optimum pH 5.0-6.0. Although β-fucosidase activities have been reported for members of GH1 (Cairns et al 2000;Harada et al 2005;Kempton and Withers 1992;Marana et al 2001), GH5 (Yoshida et al 2011), and GH42 (Benešová et al 2010) families, and β-fucosidase activity has been purified from the latex serum of Lactuca sativa (Giordani and Noat 1988), an enzyme that Amino acid sequences of DG motifs from GK3214 (GenBank accession number, BAD77499), GK3214 mutants (NFE, MRF, and M8), GK1856 (BAD76141), and GK2337 (BAD7662) were compared with related sequences of 6-phospho-β-glycosidases (Witt et al 1993) from L. lactis (AAA25173), Lactobacillus casei (ACG55692), Staphylococcus aureus, E. coli (AAA23511) (Wilson and Fox 1974), and B. subtilis (BAA08975) (Setlow et al 2004) and of β-glycosidases from P. polymyxa (AAA22263) (Sanz-Aparicio et al 1998), Bifidobacterium breve (BAA19881) (Nunoura et al 1996), S. paucimobilis (AAG59862) (Marques et al 2003), P. furiosus (AAC25555 and AAC44387) (Bauer et al 1996), S. solfataricus (NP_344331) (Corbett et al 2001), Reticulitermes flavipes (ADK12988) (Scharf et al 2010), and Dalbergia cochinchinensis (AF163097) (Cairns et al 2000). Three highly conserved amino acids in DG motifs, aspartic acid (D), arginine (R), and glycine (G) residues, are indicated by solid triangles.…”

The heterologous expression of aGlycine maxhomolog of NONEXPRESSOR OF PR1 (NPR1) and α-hydroxynitrile glucosidase suppresses parasitism by the root pathogenMeloidogyne incognitainGossypium hirsutum

“…β-Glycosidases activate β-glycosides, secondary metabolites that perform a wide array of cellular functions including the induction of symbiosis, breakdown of endosperm cell wall during germination, phytohormone activation, lignification, the production of aromatic compounds and defense (Nisius 1988;Poulton 1990;Dharmawardhana et al 1995;Leah et al 1995;Hungria & Stacey 1997;Kristoffersen et al 2000;Mizutani et al 2002;Morant et al 2003;Fia et al 2005;Maicas & Mateo 2005;Escamilla-Trevino et al 2006;Halkier & Gershenzon 2006;Lee et al 2006;Suzuki et al 2006;Naoumkina et al 2007;Stauber et al 2012). Some of the defense β-glycosides include α-hydroxynitrile glucosides, benzoxazinoid glycosides, avenacosides, isoflavonoid glycosides and glucosinolates (Niemeyer 1988;Nisius 1988;Poulton 1990;Cairns et al 2000;Halkier & Gershenzon, 2006). Among these, the α-hydroxynitrile glucosides are the best characterized, found in phylogenetically diverse taxa including ferns, gymnosperms, monocots, dicots and many agriculturally relevant plants and spanning more than 3000 plant species (Seigler & Brinker 1993;Jones 1998;Bak et al 2006;reviewed in Gleadow & Møller 2014).…”