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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

The β-Glucosidases Responsible for Bioactivation of Hydroxynitrile Glucosides in Lotus japonicus^1,[W]

Plant Biochemistry Laboratory, Department of Plant Biology, Center for Molecular Plant Physiology and VKR Research Centre "Pro-Active Plants" (A.V.M., N.B., M.E.K., C.H.K., K.J., B.L.M., S.B.), and Department of Natural Sciences (C.E.O.), University of Copenhagen, DK–1871 Frederiksberg C, Copenhagen, Denmark; Department of Biological Structure, University of Washington, Seattle, Washington 98195–7420 (S.M.P.); Centre de Recherches sur les Macromolécules Végétales, CERMAV-CNRS, FR–38041 Grenoble cedex 9, France (A.I.); and Lehrstuhl für Pflanzenphysiologie, Ruhr-Universität Bochum, D–44801 Bochum, Germany (M.P.)

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 β-glucosidases. A mixture of two hydroxynitrile glucoside-cleaving β-glucosidases was isolated from L. japonicus leaves and identified by protein sequencing as LjBGD2 and LjBGD4. The isolated hydroxynitrile glucoside-cleaving β-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 β-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.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Søren Bak (bak{at}life.ku.dk).

^[W] The online version of this article contains Web-only data.

www.plantphysiol.org/cgi/doi/10.1104/pp.107.109512

^* Corresponding author; e-mail bak{at}life.ku.dk.

Received September 28, 2007; accepted May 6, 2008; published May 8, 2008.