Catabolism of (R)-Amygdalin and (R)-Vicianin by Partially Purified ß-Glycosidases from Prunus serotina Ehrh. and Davallia trichomanoides

Abstract

Mature black cherry (Prunus serotina Ehrh.) seeds accum ulate high levels of the cyanogenic disaccharide (R)-amygdalin. Extracts from these seeds contain two β-glycosidases which have been identified and completely resolved by DEAE-cellulose ion-exchange chromatography. Amygdalin hydrolase hydrolyzed (R)-am ygdalin at an optimum pH of 5.5, releasing (R)-prunasin and D-glucose. This enzyme showed highest activity towards (R)-am ygdalin and failed to hydrolyze (R)-prunasin. linamarin, β-gentiobiose and cellobiose. A distinct β-glycosidase, prunasin hydrolase, displayed a pronounced preference for (R)-prunasin, hydrolyzing this cyanogenic monosaccharide at an optimum pH of 6.5 to mandelonitrile and D-glucose. Prunasin hydrolase was inactive towards (R)-am ygdalin, linamarin, and β-gentiobiose. Both enzymes showed significant activity towards the artificial substrates β-ONPGlu and β-PNPGlu but did not hydrolyze α-PNPGlu. In view of the pronounced specificity of these enzymes towards endogenous cyanogens, it is concluded that upon disruption of black cherry seeds (R)- amygdalin is catabolized to mandelonitrile in a stepwise manner (the sequential mechanism) by amygdalin hydrolase and prunasin hydrolase with (R)-prunasin serving as intermediate. Young fronds of Davallia trichomanoides are rich sources of (R)-vicianin (the β-vicianoside of (R)-mandelonitrile). A β-glycosidase, vicianin hydrolase, has been partially purified from frond extracts by ion-exchange chromatography. At the optimum pH of 6.0, this enzyme showed highest hydrolytic activity with (R)-vicianin, although both (R)-am ygdalin and (R)-prunasin could be hydrolyzed at approximately 15% of the rate observed with (R)-vicianin. It failed to hydrolyze β-gentiobiose, cellobiose, linamarin and α-PNPGlu. Closer exam ination revealed that (R)-vicianin and (R)-amygdalin were hydrolyzed at the aglycone-disaccharide bond (the simultaneous mechanism) yielding mandelonitrile and the respective disaccharides vicianose and β-gentiobiose

A recycling pathway for cyanogenic glycosides evidenced by the comparative metabolic profiling in three cyanogenic plant species

“…Previous reports on the presence of diglycosides suggested that the glycosides identified are probably glucose-based and predominantly exist as the -(1 6) linkage [19,21,[30][31][32]. In order to confirm this, we analyzed selected glycosides by ion-mobility MS (IM-MS), a rapid and sensitive technique that separates gas phase ions based on their mobility (inversely proportional to a molecules collision cross-section area) as they traverse through an inert buffer gas [33, for review see 34].…”

Mobilization and Utilization of Cyanogenic Glycosides

“…It can occur in one step by a simultaneous mechanism (8) producing a disaccharide and the aglycon or by the sequential mechanism (8), where the two sugars are split off from the diglucoside one after the other in two steps. It can be shown that the simultaneous but not the sequential mechanism is realized in Hevea.…”

Prunus serotina Amygdalin Hydrolase and Prunasin Hydrolase

“…the sequential action of the enzymes AH2, PH, and MDL (12,29). One of the complexities of the Prunus cyanogenic system yet to be satisfactorily unraveled is the microheterogeneity shown by these catabolic enzymes.…”

“…Monospecific antisera deemed suitable for immunocytochemistry and screening of expression libraries were obtained by affinity chromatography. Each antiserum recognized all known isozymes of the specific glucosidase used as antigen.the sequential action of the enzymes AH2, PH, and MDL (12,29). One of the complexities of the Prunus cyanogenic system yet to be satisfactorily unraveled is the microheterogeneity shown by these catabolic enzymes.…”