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

Abstract

The plant UDP-dependent glucosyltransferase (UGT) BpUGT94B1 catalyzes the synthesis of a glucuronosylated cyanidinderived 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 UDPglucose. 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.

Functional Differentiation of the Glycosyltransferases That Contribute to the Chemical Diversity of Bioactive Flavonol Glycosides in Grapevines (Vitis vinifera)

“…This conclusion corroborates the recent hypothesis that an Arg residue proximal to the carboxylate of UDP-GA is the biochemical basis of GATs of both plant and animal origin (Noguchi et al, 2009a). Arg-140 does not correspond to Arg-25 of Bp UGAT (UGT94B1) (Osmani et al, 2008) or Arg-350 of Pf F7GAT (UGT88D7) (Noguchi et al, 2009a) in primary structure, providing another example of convergent evolution of plant GATs. This illustrates the positional plasticity of the crucial Arg residue in acquiring specificity for UDP-GA in UGT catalysis (see Supplemental Figure 2 …”

“…Previous studies of two GATs, Bp UGAT (UGT94B1) of the red daisy flower (Bellis perennis) (Sawada et al, 2005;Osmani et al, 2008) and Pf F7GAT (UGT88D7) of Perilla frutescens (Noguchi et al, 2009a), showed that Arg-25 in Bp UGAT and Arg-350 in Pf F7GAT play an important role in recognition of UDP-GA. These results suggest plasticity of the mechanisms that alter the sugar donor specificity of UGTs.…”

Identification of Residues That Confer Sugar Selectivity to UDP-Glycosyltransferase 3A (UGT3A) Enzymes

“…Residues that confer UDP-GlcUA specificity have been examined using site-directed mutagenesis and protein modeling of BpUGT94B1, a UGT from red daisy (Bellis perennis) that conjugates glucuronic acid to flavonoids. This analysis identified an arginine outside of the signature sequence (Arg-25) as crucial for activity with UDP-GlcUA, with Arg-25 mutants exhibiting dramatically reduced activity with UDP-GlcUA and 3-fold increased activity with UDP-Glc (28). A conserved arginine is found in the corresponding position relative to the mature N terminus of all human UGTs of the UGT1 and UGT2 families.…”

Volatile Glycosylation in Tea Plants: Sequential Glycosylations for the Biosynthesis of Aroma β-Primeverosides Are Catalyzed by Two Camellia sinensis Glycosyltransferases

“…The sugar donor specificity of UGTs is dictated by a small number of amino acid residues (Osmani et al, 2008;Noguchi et al, 2009;Ono et al, 2010a). The residues are located in three distinct domains: N-terminal, middle, and C-terminal (PSPG box) domains (Sayama et al, 2012).…”