Flavonoids are an important group of secondary metabolites found in plants and have a wide variety of properties. Some play a role in fl ower pigmentation, while others have antimicrobial properties. Glucosylation is an important modifi cation of fl avonoids and is mediated by glucosyltransferases. In this process, the enzyme transfers glucose from UDP-glucose to a specifi c position on the fl avonoid. Previous study from the lab characterized a glucosyltransferase from C. paradisi that is fl avonol specifi c. In this study an attempt has been made to study the structure and function of this fl avonol specifi c glucosyltransferase using site directed mutagenesis. The glutamine residue at position 87 of the Cp-3-O-GT enzyme was changed to isoleucine, the analogous residue in the 3-O-glucosyltransferase of Clitoria ternatea. Similarly, the histidine at position 154 was changed to tyrosine. We hypothesize that these mutations will change substrate specifi city. The glutamate at position 88 was changed to an aspartic acid. We hypothesize that this will change the regiospecifi city of the enzyme, as aspartic acid is the analogous residue found in some 7-O-glucosyltransferases. Finally, we introduced a double mutation with glutamine 87 becoming isoleucine and glutamate 88 becoming aspartic acid, with the hypothesis that both regiospecifi city and substrate specifi city will be changed.

Citrus and other fruits produce secondary metabolites that are synthesized, regulated, and modified in part by a class of enzymes called glycosyltransferases. This class of enzymes is of substantial interest to this lab due to their unique structural and functional properties. Glycosides of flavonoids produced by glycosyltransferases have emerged in recent years as a critical part of plant metabolism, thus impacting every aspect of their growth, cultivation, production, and utilization. One such glycosyltransferase, found in Duncan Grapefruits (Citrus paradisi), was previously identified, recombinantly expressed, and shown through biochemical characterization to exclusively glycosylate the flavonol class of flavonoids. The structural basis that accounts for a glycosyltransferase's selectivity has been determined by protein crystallization in other labs, yet no structural basis currently exists for the specificity exhibited by this flavonol-specific glycosyltransferase. Currently, the WT enzyme and two mutants were expressed in E. coli, where they underwent site-directed mutagenesis to insert thrombin cleavage tags for removal of protein purification vectors, with the goal of transforming into yeast for adequate protein production. Subsequent purification and crystallization screens will allow for formation and acquisition of glycosyltransferase crystals, whose x-ray diffraction patterns will be decoded, thus revealing the enzyme's complete structure. We hypothesize that obtaining a crystal structure for this enzyme will illuminate the structural basis of its specificity. Additionally, we hypothesize that a thrombin- cleavage gene vector inserted for removal of purification tags will have no impact on enzyme activity or specificity.

Flavonoids are the C-15 phenolic compounds containing two phenyl rings and a heterocyclic ring. The majority of the flavonoids accumulated in grapefruit are flavonol, flavanone, flavone, dihydroflavonol, and chalcone glycosides. Most flavonoids are present in glucosylated form and the glucosylation is mediated by a class of enzymes called glucosyltransferases that transfer glucose from a high energy sugar donor to the acceptor aglycone at a particular position. A clone encoding a flavonol-specific 3-O-glucosyltransferase (Cp-3-O-GT) from Citrus paradisi has been previously characterized in our lab. The study of structure and function of flavonoid GTs is an important aspect of our research that contributes to the synthesis of novel glucosides by changing the glucosylation patterns of GTs. Our study focuses on the structural and functional analysis of Cp-3-O-GT through site directed mutation and analysis of mutated enzyme in terms of substrate specificity and regiospecificity. Multiple sequence alignment and homology modeling was used to identify candidate areas for mutation. For this study, Cp-3-O-GT was modeled with a flavonoid 3- O-GT from Vitis vinifera (VvGT) that can glucosylate both flavonols and anthocyanidins. We identified a proline residue at position 145 of Cp-3-O-GT that corresponded to a threonine residue in VvGT and designed a Cp-3-O-GT – P145T mutant to test the hypothesis that that mutation of key amino acid residues (proline) in Cp-3-O-GT by position specific amino acids of VvGT (threonine) could alter substrate specificity or regiospecificity of Cp-3-O-GT. Initial screening results suggested that the mutant P145T glucosylates flavanones and flavones in addition to flavonols. This is significant because flavanones and flavonols do not contain a 3-OH group for glucosylation. HPLC was performed to identify the reaction products. Early results indicate that the P145T mutant glucosylates naringenin at 7-OH position forming naringenin-7-O-glucoside and this is being confirmed. Product identification with other substrates is also being conducted. Results are being used to revisit and refine the structure model.

An outbreak of Deidamia inscriptum (Lettered Sphinx Moth) caterpillars was noted in northeast Tennessee where Oxydendrum arboreum (Sourwood) trees were defoliated. Nearly all published literature and online resources list only plants in the grape family (Vitaceae) as larval food plants. Food-plant preference trials using fresh leaves of 3 woody plant species showed that Deidamiacaterpillars from this region had a preference for Sourwood over Parthenocissus quinquefolia(Virginia Creeper), and rejected Acer rubrum (Red Maple), a non-host species. Ursus americanus(Black Bear) were feeding on the caterpillars as evidenced by bent and broken Sourwood saplings bearing claw marks and by abundant sphingid remains in bear scat.

Glucosyltransferases (GTs) are the important group of enzymes which facilitates the incorporation of UDPactivated glucose to a corresponding acceptor molecule through glucosylation. Glucosylation is a common alteration reaction in plant metabolism and is regularly associated with the production of secondary metabolites. Glucosylation serves a number of roles within metabolism including: stabilizing structures, affecting solubility, transport, and regulating the bioavailability of the compounds for other metabolic processes. GTs involved in secondary metabolism share a conserved 44 amino acid residue motif (60–80% identity) known as the plant secondary product glucosyltransferase (PSPG) box, which has been demonstrated to include the UDP-sugar binding moiety. Among the secondary metabolites, flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications in these species. Custom design of enzymes requires understanding of structure/function of the protein. The present study focuses on creating mutant Flavonol- 3-O- Glucosyltransferases proteins using site-directed mutational analysis and testing the effect of each mutation on substrate specificity and kinetic properties of the enzyme.

The wide diversity of plant secondary products results from different modifications undergone during biosynthesis, including glucosylation. These modification reactions result in production of the compounds actually found in plants and to unique chemical and biochemical properties, including some bitter compounds in grapefruit. While the presence of a PSPG box motif allows for identification of a clone as a putative glucosyltransferase (PGT), diversity of GT primary structures makes it difficult to accurately assign specific function. Our approach is to identify and isolate putative GT clones, express them heterologously, and biochemically characterize the proteins. Eleven putative GT clones have been isolated from Citrus paradise and some have been biochemically characterized. The current hypothesis being tested is that PGT3 and PGT9 clones are plant secondary product GTs. Due to issues with inclusion bodies when using E. coli, proteins were expressed in Pichia pastoris using the pPICZA vector. Recombinant protein expression was confirmed by Western blot and proteins were enriched by IMAC. Over 30 flavonoid and simple phenolic substrates, representing many compounds found in grapefruit, were screened for activity with PGT3 and PGT9 proteins. No significant activity was found and the biochemical function of the proteins encoded by these clones will be further investigated.

No description available.

Glucosyltransferases (GTs) are enzymes that enable transfer of glucose from an activated donor (UDP-glucose) to the acceptor substrates. A flavonol specific glucosyltransferase cloned from Citrus paradisi has strict substrate and regiospecificity (Cp3OGT). The amino acid sequence of Cp3OGT was aligned with a purported anthocyanin GT from Clitorea ternatea and a GT from Vitis vinifera that can glucosylate both flavonols and anthocyanidins. Using homology modeling to identify candidate regions followed by site directed mutagenesis, three double mutations of Cp3OGT were made. Biochemical analysis of the three mutant proteins was performed. S20G+T21S protein retained activity similar to the wildtype (WT- Kmapp-80 µM; Vmax = 16.5 pkat/µg, Mutant- Kmapp-83 µM; Vmax -11 pkat/µg) but the mutant was more thermostable compared to the WT and this mutation broadened its substrate acceptance to include the flavanone, naringenin. S290C+S319A mutant protein retained 40% activity relative to wildtype, had an optimum pH shift, but had no change in substrate specificity (Kmapp-18 µM; Vmax-0.5 pkat/µg). H154Y+Q87I protein was inactive with every class of flavonoid tested. Product identification revealed that the S20G+T21S mutant protein widened the substrate and regio-specificity of CP3OGT. Docking analysis revealed that H154 and Q87 could be involved in orienting the ligand molecules within the acceptor binding site. H363, S20, and S150 were also found to make close contact with the 7-OH, 4-OH and 3’-OH groups, respectively.

A flora of the 3800 ha Rocky Fork Tract in northeast Tennessee produced 749 species of which 19 were on the Tennessee Rare Plant List and 34 were on the Cherokee National Forest Species Viability List with 87 county records from Greene County and 217 from Unicoi County. Rare species were particularly numerous in the Cyperaceae and Orchidaceae. The tract serves as a refuge for several regionally uncommon species by supporting either large populations or metapopulations of these species. Exotic species comprised 15% of the flora and were most common in the Fabaceae and Poaceae. The most unique habitat was a heath bald dominated by Rhododendron catawbiense with abundant Xerophyllum asphodeloides in the herbaceous layer. While species richness was relatively high compared to regional sites of comparable area, diversity was limited by the absence of high elevation spruce-fir communities and the paucity of wetlands.

No description available.