A Kinetic Analysis of Regiospecific Glucosylation by Two Glycosyltransferases of Arabidopsis thaliana

Abstract

Plant Family 1 glycosyltransferases (GTs) recognize a wide range of natural and non-natural scaffolds and have considerable potential as biocatalysts for the synthesis of small molecule glycosides. Regiospecificity of glycosylation is an important property, given that many acceptors have multiple potential glycosylation sites. This study has used a domain-swapping approach to explore the determinants of regiospecific glycosylation of two GTs of Arabidopsis thaliana, UGT74F1 and UGT74F2. The flavonoid quercetin was used as a model acceptor, providing five potential sites for O-glycosylation by the two GTs. As is commonly found for many plant GTs, both of these enzymes produce distinct multiple glycosides of quercetin. A high performance liquid chromatography method has been established to perform detailed steady-state kinetic analyses of these concurrent reactions. These data show the influence of each parameter in determining a GT product formation profile toward quercetin. Interestingly, construction and kinetic analyses of a series of UGT74F1/F2 chimeras have revealed that mutating a single amino acid distal to the active site, Asn-142, can lead to the development of a new GT with a more constrained regiospecificity. This ability to form the 4-O-glucoside of quercetin is transferable to other flavonoid scaffolds and provides a basis for preparative scale production of flavonoid 4-O-glucosides through the use of wholecell biocatalysis.The glycosylation of small hydrophobic molecules is known to alter their chemical and biological characteristics. The alterations include changes in water solubility, stability, pharmacokinetic properties, and bioactivity (1, 2). Enzymes that catalyze these reactions are Family 1 glycosyltransferases (GTs), 3 with representatives found in a wide range of prokaryotic and eukaryotic organisms.Extensive study of GT activities has demonstrated that the enzymes display an exquisite regio-(3), enantio-(4), and chemospecificity (5) toward the acceptor molecule. However, as yet the determinants of this specificity are poorly understood. This lack of understanding presents challenges to the interpretation of substrate activity data (6, 7), GT rational redesign (8, 9), and activity prediction (10, 11). Future progress in predicting GT sequence-structure-activity relationships will depend on a greater number of studies that characterize the key determinants of activity and specificity.Quercetin is the most abundant flavonoid in the human diet (12) and has frequently been used as a model substrate for GT activity (13-15). Quercetin aglycone is found only at low concentrations in the primary dietary source, plants, where glycosylated forms predominate (16). In addition, in mammals, glycosides are the major byproducts of quercetin phase II metabolism (17). Many studies have shown that glycosylation of quercetin significantly affects bioavailability and efficacy with respect to anti-oxidant, anti-proliferative, and anti-cancer properties (18). There are also some data to indicate that the positi...

Novel Plant Immune-Priming Compounds Identified via High-Throughput Chemical Screening Target Salicylic Acid Glucosyltransferases in Arabidopsis

“…However, the avenalumin structure had been corrected as avenanthramide (Miyagawa et al, 1995), and Imprimatin A is not similar to avenanthramide. Just as quercetin serves as an artificial substrate for UGT74F1 (Cartwright et al, 2008), Imprimatins also might be glucosylated by SAGTs, resulting in perturbation of SA metabolism by competition. Further structure-activity relationship analyses or drug metabolism studies in planta will reveal the core motif required for activity.…”

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 changes were explained by changed size of the acceptor pocket resulting from introduction of smaller amino acid side chains, allowing for different positioning of the acceptor molecules (He et al, 2006). Change in the size of a single amino acid residue has also been observed to alter acceptor specificity of other plant UGTs (Masada et al, 2007;Cartwright et al, 2008). Eight other point mutations in the acceptor pocket of MtUGT71G1 resulted in activities of 20% to 90% of wild-type protein (He et al, 2006).…”

“…It also implies that novel combinations of donor and acceptor specificity might be obtained by combining entire N-and C-terminal domains of different UGTs. In recent studies (Cartwright et al, 2008;Weis et al, 2008) it has been possible to construct such functionally active chimeric UGTs that harbor the N-and C-terminal domains from different plant UGTs.…”

Characterization of a Glucosyltransferase Enzyme Involved in the Formation of Kaempferol and Quercetin Sophorosides in Crocus sativus

“…Furthermore, several different Escherichia coli strains were also tested without success. Thus, whole-cell biotransformation was carried out according to Cartwright et al (2008), but again, we were unable to detect any activity.…”

“…Whole-cell biotransformations were performed as described previously (Cartwright et al, 2008) with minor modifications. In brief, E. coli Rossetta (LysS) cells harboring UGT707B1 in a pGEX-5X-3 vector were grown at 28°C, 150 rpm, in 200 mL of M9 minimal medium containing 0.4% glycerol, 100 mg mL 21 AMP, and 60 mg mL 21 CM to A 600 = 0.75 and then induced with 0.1 mM isopropylthio-b-galactoside and incubated for 16 h. After the induction period, each substrate, kaempferol, quercetin, apigenin, naringenin, caffeic acid, and luteolin, was added to the bacterial cultures with a final concentration of 50 mM.…”