The beta-glucosidases responsible for bioactivation of hydroxynitrile glucosides in Lotus japonicus.
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 beta-glucosidases. A mixture of two hydroxynitrile glucoside-cleaving beta-glucosidases was isolated from L. japonicus leaves and identified by protein sequencing as LjBGD2 and LjBGD4. The isolated hydroxynitrile glucoside-cleaving beta-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 beta-glucosidase, LjBGD7. Reverse transcription-polymerase 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.
Morant
Anne Vinther
AV
Plant Biochemistry Laboratory, Department of Plant Biology, Center for Molecular Plant Physiology and VKR Research Centre "Pro-Active Plants" , University of Copenhagen, DK-1871 Frederiksberg C, Copenhagen, Denmark.
Bjarnholt
Nanna
N
Kragh
Mads Emil
ME
Kjaergaard
Christian Hauge
CH
Jørgensen
Kirsten
K
Paquette
Suzanne Michelle
SM
Piotrowski
Markus
M
Imberty
Anne
A
Olsen
Carl Erik
CE
Møller
Birger Lindberg
BL
Bak
Søren
S
D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites; D044602:Cellulases / Q000235:genetics / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005960:Glucosides / Q000378:metabolism*; D006868:Hydrolysis; D007527:Isoenzymes / Q000378:metabolism; D000070116:Lotus / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D009281:Naphthalenes / Q000378:metabolism; D009570:Nitriles / Q000378:metabolism*; D010802:Phylogeny; D018515:Plant Leaves / Q000378:metabolism*; D018517:Plant Roots / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D005960:Glucosides; D007527:Isoenzymes; D009281:Naphthalenes; D009570:Nitriles; D003577:Cytochrome P-450 Enzyme System; C492216:cytochrome P-450 79D3, Lotus japonicus; D044602:Cellulases; C001556:lotaustralin