News
September 17th, 2009
Substrate specificity of plant UDP-dependent glycosyltransferases predicted from crystal structures and homology modeling
Plant family 1 UDP-dependent glycosyltransferases (UGTs) catalyze the glycosylation of a plethora of bioactive
natural products. In Arabidopsis thaliana, 120 UGT encoding genes have been identified. The crystal-
based 3D structures of four plant UGTs have recently been published. Despite low sequence
conservation, the UGTs show a highly conserved secondary and tertiary structure. The sugar acceptor
and sugar donor substrates of UGTs are accommodated in the cleft formed between the N- and C-terminal
domains. Several regions of the primary sequence contribute to the formation of the substrate binding
pocket including structurally conserved domains as well as loop regions differing both with respect to
their amino acid sequence and sequence length. In this review we provide a detailed analysis of the available
plant UGT crystal structures to reveal structural features determining substrate specificity. The high
3D structural conservation of the plant UGTs render homology modeling an attractive tool for structure
elucidation. The accuracy and utility of UGT structures obtained by homology modeling are discussed and
quantitative assessments of model quality are performed by modeling of a plant UGT for which the 3D
crystal structure is known. We conclude that homology modeling offers a high degree of accuracy. Shortcomings
in homology modeling are also apparent with modeling of loop regions remaining as a particularly
difficult task.
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March 10th, 2008
Catalytic Key Amino Acids and UDP-Sugar Donor Specificity of a Plant Glucuronosyltransferase, UGT94B1: Molecular Modeling Substantiated by Site-Specific Mutagenesis and Biochemical Analyses
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.
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April 4th, 2008
During the first year of the CAPPELLA project, the consortium have successfully established functional protein:protein interaction (PPI) assays for 3 important cancer targets. Both targets implicated in a broad range of tumors and targets particularly relevant to colon and breast cancer are being investigated. Primary screening for anti-cancer compounds targeting these PPIs have been initiated and a number of promising inhibitory small molecule compounds have been identified in all 3 assays.