Received September 28, 2007
Accepted May 6, 2008
The 
-glucosidases responsible for bio-activation of hydroxynitrile glucosides in Lotus japonicus
Anne Vinther Morant , Nanna Bjarnholt , Mads Emil Kragh , Christian Hauge Kjargaard , Kirsten Jorgensen , Suzanne Michelle Paquette , Markus Piotrowski , Anne Imberty , Carl Erik Olsen , Birger Lindberg Moller , and Soren Bak *
Plant Biochemistry Laboratory, Department of Plant Biology, Center for Molecular Plant Physiology and VKR centre of excellence "Pro-Active Plants" and Department of Natural Sciences, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark; Department of Biological Structure, University of Washington, HSB G-514/Box 357420, Seattle, WA 98195-7420, USA; Centre de Recherches sur les Macromolecules Vegetales (CERMAV-CNRS), BP 53, FR-38041 Grenoble cedex 9, France (Affiliated with the Joseph Fourier University of Grenoble and member of the Institut de Chimie Moleculaire de Grenoble) and Lehrstuhl fur Pflanzenphysiologie, Ruhr-Universitat Bochum, Universitatsstrase 150, D-44801 Bochum, Germany
* Corresponding author; email: bak{at}life.ku.dk.
Lotus japonicus accumulates the hydroxynitrile glucosides lotaustralin, linamarin and rhodiocyanosides A and D. Upon tissue disruption, the hydroxynitrile glucosides are bio-activated by hydrolysis by specific 
-glucosidases. A mixture of two hydroxynitrile glucoside cleaving -glucosidases was isolated from L. japonicus leaves and identified by protein sequencing as LjBGD2 and LjBGD4. The isolated hydroxynitrile glucoside cleaving -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 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 -glucosidase, LjBGD7. RT-PCR 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 biosyntheis of hydroxynitrile glucosides in L. japonicus, with CYP79D3 expression in aerial tissues and CYP79D4 expression in roots.
