title: 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 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.
authors.affiliation: VKR Research Centre Pro-Active Plants, DK-1871 Frederiksberg C, Copenhagen, Denmark.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Osmani
authors.forename: Søren
authors.initials: S
authors.lastname: Bak
authors.forename: Anne
authors.initials: A
authors.lastname: Imberty
authors.forename: Carl Erik
authors.initials: CE
authors.lastname: Olsen
authors.forename: Birger Lindberg
authors.initials: BL
authors.lastname: Møller
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D001483:Base Sequence; D055162:Biocatalysis; D017931:DNA Primers; D014453:Glucuronosyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014539:Uridine Diphosphate Sugars / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D017931:DNA Primers; D014539:Uridine Diphosphate Sugars; D014453:Glucuronosyltransferase