A single amino acid in the PSPG‐box plays an important role in the catalytic function of CaUGT2 (Curcumin glucosyltransferase), a Group D Family 1 glucosyltransferase from <i>Catharanthus roseus</i>

Masada, Sayaka; Terasaka, Kazuyoshi; Mizukami, Hajime

doi:10.1016/j.febslet.2007.05.002

Cited by 31 publications

(20 citation statements)

References 36 publications

(52 reference statements)

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“…By domain swapping between two GTs of 85% identity, Brazier-Hicks et al (5) found a region of five variable amino acids responsible for enabling the formation of N-glucosides in addition to O-glucosides. Other studies (30,31) found that at lower levels of sequence identity (75 and 28%, respectively) the generated chimeras were inactive.…”

Section: Discussionmentioning

confidence: 99%

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

Cartwright

Lim

Kleanthous

et al. 2008

Journal of Biological Chemistry

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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...

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Section: Discussionmentioning

confidence: 99%

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

Cartwright

Lim

Kleanthous

et al. 2008

Journal of Biological Chemistry

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“…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).…”

Section: Sugar Acceptor Specificity Of Bpugt94b1mentioning

confidence: 99%

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

et al. 2008

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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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“…A previous study reported that a nonconserved residue, Cys-377, in the PSPG-box of CaUGT2 plays an essential role in determining the catalytic function of CaUGT2 (Masada et al, 2007). In this study, we have shown that the D343E, K458R, and D445E single mutants and the G433A/I434V double mutant of UGT71A33 all displayed enhanced HDMF glucosylation activity, particularly the D343E mutant and G433A/I434V double mutant, which exhibited increases in activity more than 2-fold above that of the wild-type enzyme UGT71A33 (Fig.…”

Section: Site-directed Mutagenesis Identified Residues Important For mentioning

confidence: 99%

Glucosylation of 4-Hydroxy-2,5-Dimethyl-3(2H)-Furanone, the Key Strawberry Flavor Compound in Strawberry Fruit

et al. 2016

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ORCID IDs: 0000-0002-7852-1194 (C.S.); 0000-0002-9753-3967 (W.S.).Strawberries emit hundreds of different volatiles, but only a dozen, including the key compound HDMF [4-hydroxy-2,5-dimethyl-3(2H)-furanone] contribute to the flavor of the fruit. However, during ripening, a considerable amount of HDMF is metabolized to the flavorless HDMF b-D-glucoside. Here, we functionally characterize nine ripening-related UGTs (UDPglucosyltransferases) in Fragaria that function in the glucosylation of volatile metabolites by comprehensive biochemical analyses. Some UGTs showed a rather broad substrate tolerance and glucosylated a range of aroma compounds in vitro, whereas others had a more limited substrate spectrum. The allelic UGT71K3a and b proteins and to a lesser extent UGT73B24, UGT71W2, and UGT73B23 catalyzed the glucosylation of HDMF and its structural homolog 2(or 5)-ethyl-4-hydroxy-5(or 2)-methyl-3(2H)-furanone. Site-directed mutagenesis to introduce single K458R, D445E, D343E, and V383A mutations and a double G433A/I434V mutation led to enhanced HDMF glucosylation activity compared to the wild-type enzymes. In contrast, a single mutation in the center of the plant secondary product glycosyltransferase box (A389V) reduced the enzymatic activity. Down-regulation of UGT71K3 transcript expression in strawberry receptacles led to a significant reduction in the level of HDMF-glucoside and a smaller decline in HDMF-glucoside-malonate compared with the level in control fruits. These results provide the foundation for improvement of strawberry flavor and the biotechnological production of HDMF-glucoside.

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A single amino acid in the PSPG‐box plays an important role in the catalytic function of CaUGT2 (Curcumin glucosyltransferase), a Group D Family 1 glucosyltransferase from Catharanthus roseus

Cited by 31 publications

References 36 publications

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

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

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

Glucosylation of 4-Hydroxy-2,5-Dimethyl-3(2H)-Furanone, the Key Strawberry Flavor Compound in Strawberry Fruit

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