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

Abstract

Lotus japonicus accumulates the hydroxynitrile glucosides lotaustralin, linamarin, and rhodiocyanosides A and D. Upon tissue disruption, the hydroxynitrile glucosides are bioactivated by hydrolysis by specific b-glucosidases. A mixture of two hydroxynitrile glucoside-cleaving b-glucosidases was isolated from L. japonicus leaves and identified by protein sequencing as LjBGD2 and LjBGD4. The isolated hydroxynitrile glucoside-cleaving b-glucosidases preferentially hydrolyzed rhodiocyanoside A and lotaustralin, whereas linamarin was only slowly hydrolyzed, in agreement with measurements of their rate of degradation upon tissue disruption in L. japonicus leaves. Comparative homology modeling predicted that LjBGD2 and LjBGD4 had nearly identical overall topologies and substrate-binding pockets. Heterologous expression of LjBGD2 and LjBGD4 in Arabidopsis (Arabidopsis thaliana) enabled analysis of their individual substrate specificity profiles and confirmed that both LjBGD2 and LjBGD4 preferentially hydrolyze the hydroxynitrile glucosides present in L. japonicus. Phylogenetic analyses revealed a third L. japonicus putative hydroxynitrile glucoside-cleaving b-glucosidase, LjBGD7. Reverse transcriptionpolymerase chain reaction analysis showed that LjBGD2 and LjBGD4 are expressed in aerial parts of young L. japonicus plants, while LjBGD7 is expressed exclusively in roots. The differential expression pattern of LjBGD2, LjBGD4, and LjBGD7 corresponds to the previously observed expression profile for CYP79D3 and CYP79D4, encoding the two cytochromes P450 that catalyze the first committed step in the biosyntheis of hydroxynitrile glucosides in L. japonicus, with CYP79D3 expression in aerial tissues and CYP79D4 expression in roots.

Comparative Analysis of Benzoxazinoid Biosynthesis in Monocots and Dicots: Independent Recruitment of Stabilization and Activation Functions

“…A signal peptide for targeting the proteins into the secretory pathway is predicted for both enzymes, and transit peptide sequences for localization to the plastid are not detected (TargetP 1.1, Emanuelsson et al, 2000;iSPORT, Bannai et al, 2002). The enzymes have ;45% amino acid identity to the query sequences Zm-GLU1 and At-TGG1 and ;60% amino acid identities to the closest homologous enzymes with defined function, the exoglucanase Os-4BGLU12 (Opassiri et al, 2006) and the hydroxynitril-glucoside hydrolyzing enzyme Lj-BGD2 (Morant et al, 2008a;Takos et al, 2010). Expression of the b-GLUs in E. coli failed probably due to instability caused by lack of plant-specific protein modification (Zhou et al, 2002;Morant et al, 2008b).…”

“…The enzyme mixture of the purified b-GLUs Lj-BGD2 and Lj-BGD4 that are involved in activation of cyanogenic and noncyanogenic hydroxynitril glucosides in Lotus japonicas (Takos et al, 2010) hydrolyze the isoflavonoid-glucoside daidzin (Morant et al, 2008a), although isoflavonoids are not natural substrates for these enzymes. Based on phylogenetic analysis and the promiscuity of the b-GLUs, it has been hypothesized that isoflavonoid glucosidecleaving enzymes have evolved from cyanogenic glucoside activating b-GLUs (Chuankhayan et al, 2007;Morant et al, 2008a). Parallels exist for benzoxazinoids: Cyanogenic glucosides and benzoxazinoids are found in grasses and in Ranunculaceae (Sharples et al, 1972;Vetter, 2000).…”

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

“…perform a prominent role in defense (reviewed in Jahn and Fasshauer 2012). Bioinformatics analyses show that Gm-βg-4 is most closely related to the root-specific Lotus japonicus α-hydroxynitrile glucosidase LjBGD7, belonging to a small family of enzymes involved in the production of cyanogenic α-hydroxynitrile glucosides (Morant et al 2008;Takos et al 2010). In the α-hydroxynitrile glucoside metabolic pathway, active α-hydroxynitrile glucosides are produced through a pathway involving cytochrome p450 79 D4 (CYP79D4) which converts an amino acid to an oxime.…”

ATIVIDADE DE SS-Glucosidases EM EXTRATO ENZIMÁTICO OBTIDO DE AMÊNDOAS DE PÊSSEGO

“…A hidrólise da amigdalina ocorre primeiramente pela ação de amigdalina hidrolase, que age sobre a ligação β 1-6 entre as duas glicoses. A molécula de prunassina liberada consiste no substrato para a enzima prunassina hidrolase, produzindo uma molécula de glicose e uma de mandelonitrila, que pode ser dissociada espontaneamente ou pela ação da enzima mandelonitrila liase, liberando íons cianeto (MORANT et al, 2008b). O cianeto possui potencial tóxico porque inativa o complexo citocromo oxidase da respiração celular (MORANT et al, 2008a).…”

“…a enzima prunassina hidrolase, encontrada em amêndoas amargas e doces, está localizada em vesículas específicas presentes em células do endosperma, nucelo e tegumento (SánCHeZ-PEREZ et al, 2012), separadas dos glicosídeos cianogênicos, estocados no vacúolo celular (MORANT et al, 2008a). Ocorrendo a ruptura celular, os glicosídeos cianogênicos entram em contato com β-glucosidades, o que ocasiona a hidrólise destes compostos (MORANT et al, 2008b).…”