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

Sarah A Osmani,Søren Bak,Carl Erik Olsen,Anne Imberty,Birger Lindberg Møller

doi:10.1104/pp.108.128256

Abstract

The plant UDP-dependent glucosyltransferase (UGT) BpUGT94B1 catalyzes the synthesis of a glucuronosylated cyanidin-derived flavonoid in red daisy (Bellis perennis). 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 region was identified as crucial for the activity with UDP-glucuronic acid. The mutants R25S, R25G, and R25K all exhibited only 0.5% to 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 toward delphinidin-3-O-glucoside was only 5% to 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 toward delphinidin-3-O-glucoside. In all three mutant enzymes, the enzymatic activity toward 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 plant secondary product glycosyltransferase motif, determining sugar donor and acceptor specificity.

Highlights

The plant UDP-dependent glucosyltransferase (UGT) BpUGT94B1 catalyzes the synthesis of a glucuronosylated cyanidinderived flavonoid in red daisy (Bellis perennis)
This means that high homology of the query sequence with the templates is essential to achieve reliable predictions. It underscores the need for substantial additional amounts of biochemical and structural data to enable development of bioinformatic programs with reliable predictive power. This is especially important for the GTs, as annotations based on primary structure analysis have proven difficult, and substrate specificity can be highly diverse for UGTs within groups defined by phylogenetic analyses (Richman et al, 2005; Modolo et al, 2007)
The secondary structure predictions were used for an alignment of BpUGT94B1 with sequences of the plant enzymes MtUGT71G1 (Shao et al, 2005) and VvGT1 (Offen et al, 2006; Fig. 2), for which crystal structure coordinates have been published

Summary

Introduction

The plant UDP-dependent glucosyltransferase (UGT) BpUGT94B1 catalyzes the synthesis of a glucuronosylated cyanidinderived flavonoid in red daisy (Bellis perennis). State of the art may be exemplified by the recent development of a program to predict acceptors for bacterial GTs glycosylating antibiotics (Kamra et al, 2005) This program uses a knowledgebased approach and relies on available biochemical and crystal structure data. It underscores the need for substantial additional amounts of biochemical and structural data to enable development of bioinformatic programs with reliable predictive power This is especially important for the GTs, as annotations based on primary structure analysis have proven difficult, and substrate specificity can be highly diverse for UGTs within groups defined by phylogenetic analyses (Richman et al, 2005; Modolo et al, 2007). No conserved amino acid residues have been identified as general determinants of sugar specificity (Ouzzine et al, 2002; Kubo et al, 2004; Modolo et al, 2007; Yonekura-Sakakibara et al, 2007)

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Results

Conclusion