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Published on October 1, 2008; 10.1104/pp.108.128256

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Received August 20, 2008
Accepted September 19, 2008

Catalytic Key Amino Acids and UDP Sugar Donor Specificity of a Plant Glucuronosyl Transferase, UGT94B1. Molecular Modeling Substantiated by Site-Specific Mutagenesis and Biochemical Analyses

Sarah A. Osmani , Soren. Bak , Anne Imberty , Carl Erik. Olsen , and Birger Lindberg Moller *

University of Copenhagen, Department of Plant Biology and Biotechnology, Plant Biochemistry Laboratory, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark; VKR Research Centre "Pro-Active Plants", 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark; Centre de Recherches sur les Macromolecules Vegetales, CNRS, BP53, 38041 Grenoble Cedex 09, France; University of Copenhagen, Department of Natural Sciences, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark

* Corresponding author; email: blm{at}life.ku.dk.

The plant UDP-dependent glucosyltransferase (UGT) BpUGT94B1 catalyzes the synthesis of a glucuronosylated cyanidin-derived flavonoid in Bellis perennis (Red Daisy). The functional properties of BpUGT94B1 were investigated using protein modeling, site directed mutagenesis and analysis of the substrate specificity of isolated wild type and mutated forms of BpUGT94B1. A single unique arginine residue (R25) positioned outside the conserved plant secondary product glycosyltransferase (PSPG) region was identified as crucial for the activity with UDP-glucuronic acid. The mutants R25S, R25G, and R25K all exhibited only 0.5-2.5% of wild type activity with UDP-glucuronic acid, but showed a 3-fold increase in activity with UDP-glucose. The model of BpUGT94B1 also enabled identification of key residues in the acceptor pocket. The mutations N123A and D152A decreased the activity with cyanidin 3-O-glucoside to less than 15% of wild type. The wild type enzyme activity towards delphinidin-3-O-glucoside was only 5-10% of the activity with cyanidin 3-O-glucoside. Independent point mutations of three residues positioned near the acceptor B-ring were introduced to increase the activity towards delphinidin-3-O-glucoside. In all 3 mutant enzymes the enzymatic activity towards both acceptors was reduced to less than 15% of wild type. The model of BpUGT94B1 allowed for correct identification of catalytically important residues, within as well as outside the PSPG motif, determining sugar donor and acceptor specificity.




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