Plant Physiol. Illumina
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH
QUICK SEARCH:   [advanced]


     

Plant Physiology Preview
Published on September 16, 2005; 10.1104/pp.105.063842

This Article
Right arrow Full Text (Plant Physiology Preview (PDF))
Right arrow All Versions of this Article:
139/2/664    most recent
pp.105.063842v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Thorsøe, K. S.
Right arrow Articles by Møller, B. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Thorsøe, K. S.
Right arrow Articles by Møller, B. L.
Agricola
Right arrow Articles by Thorsøe, K. S.
Right arrow Articles by Møller, B. L.

Received April 5, 2005
Returned for revision June 13, 2005
Accepted July 18, 2005

Determination of Catalytic Key Amino Acids and UDP Sugar Donor Specificity of the Cyanohydrin Glycosyltransferase UGT85B1 from Sorghum bicolor. Molecular Modeling Substantiated by Site-Specific Mutagenesis and Biochemical Analyses

Karina Sinding Thorsøe , Søren Bak *, Carl Erik Olsen , Anne Imberty , Christelle Breton , and Birger Lindberg Møller

Plant Biochemistry Laboratory, Department of Plant Biology, Royal Veterinary and Agricultural University, DK-1871 Frederiksberg C, Copenhagen, Denmark; Center for Molecular Plant Physiology, Royal Veterinary and Agricultural University, DK-1871 Frederiksberg C, Copenhagen, Denmark
Department of Natural Sciences, Royal Veterinary and Agricultural University, DK-1871 Frederiksberg C, Copenhagen, Denmark; Center for Molecular Plant Physiology, Royal Veterinary and Agricultural University, DK-1871 Frederiksberg C, Copenhagen, Denmark
Centre de Recherches sur les Macromolécules Végétales, Centre National de la Recherche Scientifique (affiliated with Université Joseph Fourier), 38041 Grenoble cedex 9, France

* Corresponding author; email: bak{at}kvl.dk.

Plants produce a plethora of structurally diverse natural products. The final step in their biosynthesis is often a glycosylation step catalyzed by a family 1 glycosyltransferase (GT). In biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor, the UDP-glucosyltransferase UGT85B1 catalyzes the conversion of p-hydroxymandelonitrile into dhurrin. A structural model of UGT85B1 was built based on hydrophobic cluster analysis and the crystal structures of two bacterial GTs, GtfA and GtfB, which each showed approximately 15% overall amino acid sequence identity to UGT85B1. The model enabled predictions about amino acid residues important for catalysis and sugar donor specificity. p-Hydroxymandelonitrile and UDP-glucose (Glc) were predicted to be positioned within hydrogen-bonding distance to a glutamic acid residue in position 410 facilitating sugar transfer. The acceptor was packed within van der Waals distance to histidine H23. Serine S391 and arginine R201 form hydrogen bonds to the pyrophosphate part of UDP-Glc and hence stabilize binding of the sugar donor. Docking of UDP sugars predicted that UDP-Glc would serve as the sole donor sugar in UGT85B1. This was substantiated by biochemical analyses. The predictive power of the model was validated by site-directed mutagenesis of selected residues and using enzyme assays. The modeling approach has provided a tool to design GTs with new desired substrate specificities for use in biotechnological applications. The modeling identified a hypervariable loop (amino acid residues 156-188) that contained a hydrophobic patch. The involvement of this loop in mediating binding of UGT85B1 to cytochromes P450, CYP79A1, and CYP71E1 within a dhurrin metabolon is discussed.




This article has been cited by other articles:


Home page
 Plant Physiol.Home page
A. Rahier, M. Bergdoll, G. Genot, F. Bouvier, and B. Camara
Homology Modeling and Site-Directed Mutagenesis Reveal Catalytic Key Amino Acids of 3{beta}-Hydroxysteroid-Dehydrogenase/C4-Decarboxylase from Arabidopsis
Plant Physiology, April 1, 2009; 149(4): 1872 - 1886.
[Abstract] [Full Text] [PDF]

Home page
 Plant Physiol.Home page
S. A. Osmani, S. Bak, A. Imberty, C. E. Olsen, and B. L. Moller
Catalytic Key Amino Acids and UDP-Sugar Donor Specificity of a Plant Glucuronosyltransferase, UGT94B1: Molecular Modeling Substantiated by Site-Specific Mutagenesis and Biochemical Analyses
Plant Physiology, November 1, 2008; 148(3): 1295 - 1308.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. M. Cartwright, E.-K. Lim, C. Kleanthous, and D. J. Bowles
A Kinetic Analysis of Regiospecific Glucosylation by Two Glycosyltransferases of Arabidopsis thaliana: DOMAIN SWAPPING TO INTRODUCE NEW ACTIVITIES
J. Biol. Chem., June 6, 2008; 283(23): 15724 - 15731.
[Abstract] [Full Text] [PDF]

Home page
 Plant Physiol.Home page
Y. H. Gebhardt, S. Witte, H. Steuber, U. Matern, and S. Martens
Evolution of Flavone Synthase I from Parsley Flavanone 3beta-Hydroxylase by Site-Directed Mutagenesis
Plant Physiology, July 1, 2007; 144(3): 1442 - 1454.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH
ASPB Publications PLANT PHYSIOLOGY® THE PLANT CELL
Copyright © 2005 by the American Society of Plant Biologists