Glucosyltransferases (GTs) are enzymes that expedite the incorporation of UDP-activated glucose to a corresponding acceptor molecule. This enzymatic reaction stabilizes structures and affects solubility, transport, and bioavailability of flavonoids for other metabolic processes. Flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications. Custom design of enzymes requires understanding of structure/function of the protein. The present study focuses on creating mutant flavonol-3-O-glucosyltransferase (F-3-O-GT) proteins using site-directed mutagenesis and testing the effect of each mutation on substrate specificity, regiospecificity and kinetic properties of the enzyme. Mutations were selected on the basis of sequence similarity between grapefruit F-3-O-GT, an uncharacterized GT gene in blood orange (98%), and grape F3GT (82%). Grapefruit F-3-O-GT prefers flavonol as a substrate whereas the blood orange sequence is annotated to be a flavonoid 3GT and the grape GTs could glucosylate both flavonols and anthocyanidins. Mutants of F-3-O-GT were generated by substituting L41M, N242K, E296K and N242K+E296K and proteins were expressed in Pichia pastoris using the pPICZA vector. Analysis of these mF-3-O-GTs showed that all of them preferred flavonols over flavanone, flavone, isoflavones, or anthocyanidin substrates and showed decrease in enzyme activity of 16 to 51% relative to the wild type F-3-O-GT.

Flavonoids are a class of secondary metabolites, the majority of which are present in glucosylated form. Glucosyltransferases catalyze glucosylation by transferring glucose from UDP-activated sugar donor to the acceptor substrates. This research is focused on the study of the effect of a single point mutation on enzyme activity, characterization of a flavonol specific 3-O-glucosyltransferase (Cp-3-O-GT) mutant- P145T, and further modification of the clone to cleave off tags from recombinant wild type and P145T mutant proteins in order to crystallize the proteins. Multiple sequence alignment and homology modeling was done to identify candidate residues for mutation. 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-GTP145T mutant to test the hypothesis that that mutation of proline by threonine in Cp-3-O-GT could alter substrate or regiospecificity of Cp-3-O-GT. While the mutant P145T enzyme did not glucosylate anthocyanidins, it did glucosylate flavanones and flavones in addition to flavonols. This is significant because flavanones and flavones do not contain a 3-OH group. HPLC was performed to identify the reaction products. Early results indicated that the mutant protein glucosylates naringenin at the 7-OH position forming prunin. Results are being used to revisit and refine the structure model. In other related work, a thrombin cleavage site was inserted into wild type and recombinant P145Tenzyme and we are currently working on transformation into yeast for recombinant protein expression. Cleaving off tags is a pre-requisite to future efforts to crystallize the proteins. Solving the crustal structures will make a significant contribution to the structural and functional study of plant flavonoid GTs in general and Cp-3- O-GT in particular.

Glucosyltransferases (GTs) are enzymes that expedite the incorporation of UDP-activated glucose to a corresponding acceptor molecule. This enzymatic reaction stabilizes structures and affects solubility, transport, and bioavailability of flavonoids for other metabolic processes. Flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications. Custom design of enzymes requires understanding of structure/function of the protein. The present study focuses on creating mutant flavonol-3-O-glucosyltransferase (F-3-O-GT) proteins using site directed mutagenesis and testing the effect of each mutation on substrate specificity, regiospecificity and kinetic properties of the enzyme. Mutations were selected on the basis of sequence similarity between grapefruit F-3- O-GT, an uncharacterized GT gene in blood orange (98%), and grape F3GT (82%). Grapefruit F-3-O-GT prefers flavonol as a substrate whereas the blood orange sequence is annotated to be a flavonoid 3GT and the grape GTs could glucosylate both flavonols and anthocyanidins. Mutants of F-3-O-GT were generated by substituting N242K, E296K and N242K+E296K and proteins were expressed in Pichia pastoris using the pPICZA vector. Analysis of these mF-3-O-GTs showed that all of them preferred flavonols over flavanone, flavone, isoflavones, or anthocyanidin substrates and showed decrease in enzyme activity of 16 to 51% relative to the wild type F-3- O-GT.

Most naturally-occurring fl avonoids are found in glucosylated form. Glucosyltransferases (GTs) are enzymes that catalyze the transfer of glucose from a high energy sugar donor to an acceptor molecule. Citrus paradisi fl avonol-specifi c glucosyltransferase (Cp-F3-O-GT) is recognized for its rigid substrate and regiospecifi city. In this work, homology modeling, site-directed mutagenesis, and biochemical analyses of the recombinant mutant Cp-F3-O-GT proteins were used to investigate potential amino acid residues that might be responsible for the enzymes strict regiospecifi city while also investigating its substrate specifi city. The single point mutations of three amino acid residues within the grapefruit F3-O-GT identifi ed through sequence alignment and homology modeling were performed. Analyses of the enzyme activity of the recombinant mutant F3-O-GT proteins revealed that the single point mutations of serine 20 to leucine (S20L) and proline 297 to phenylalanine (P297F) rendered the recombinant enzymes inactive with fl avonol substrates at 6% and 12% respectively relative to wild-type. However, the mutation of glycine 392 to glutamate (G392E) remained active and glucosylated the fl avonol acceptors quercein (Km app= 11 μM; Vmax = 5.7 pKat/μg) relative to the wild-type (Km app= 93 μM; Vmax = 41.7 pKat/μg), and kaempferol (Km app= 7 μM; Vmax = 3.8 pKat/μg) relative to the wild-type (Km app = 39 μM; Vmax = 4.2 pKat/ μg). The mutant enzyme also did not show broadened acceptor substrate specifi city as it also favored fl avonols as the preferred acceptor substrate. The optimum pH of the mutant enzyme was 8.0 similar to the wild-type F3-O-GT. Activity of the mutant enzyme was stimulated by NaCl and KCl, but inhibited by Cu2+, Zn2+, Fe2+ as well as UDP with an apparent Ki of 10μM. Product identifi cation to determine glucosylation position is being investigated for a possible change in regiospecifi city.

Flavonoids are a group of plant secondary metabolites that are vital to the cell systems of plants. The intake of these chemicals is advantageous to animals for their antioxidant properties that affect the function of immune and inflammatory cells. The bitter taste of grapefruit (Citrus paradisi) and other citrus species is caused by the accumulation of glycosylated flavonoids. Glucosyltransferases (GTs) are enzymes that add glucose moieties to a carbon or hydroxyl group of natural products. The function of a putative secondary product GT clone was tested. In previous research, putative GT 4 was cloned into a pCD1 modified pET expression system, heterologously expressed in E.coli, and screened for activity with a few substrates; little GT activity was found. Issues of protein localized to inclusion bodies in bacteria were addressed. PGT 4 is being heterologously expressed in yeast (Pichia pastoris) to allow for protein production and analysis. PGT 4 was screened for GT activity with different flavonoid subclass representatives and simple phenolics.

Plant secondary products, which include the flavonoids, have a variety of roles in plant systems. Their roles include biosignalling, UV protection, antifeedant activity, pollinator attraction, stress response, and many others. Glucosylation is an important modification of many flavonoids and other plant secondary products. In grapefruit, glucosylation is important in the synthesis of the bitter compound naringin. Glucosyltransferases catalyze glucosylation reactions. Putative plant secondary product glucosyltransferases may be identified by the loosely conserved “PSPG box” amino acid sequence; however, with current knowledge, biochemical characterization is the only way to determine with certainty the function of these enzymes. The hypothesis tested here is that PGT11 is a plant secondary product glucosyltransferase. Recombinant PGT11 has been expressed in yeast using the pPICZ A vector. To investigate the hypothesis, the enzyme will be screened for glucosylation activity with various flavonoid and phenolic substrates.

Plants produce a vast array of secondary metabolites. The phenolic compounds flavonoids are metabolites ubiquitous among plants and are known to aid in processes such as plant reproduction, UV defense, pigmentation and development. In relation to human health, flavonoids have also been found to possess anti-inflammatory, anti-cancer, and anti-oxidant properties. Flavonoids ability to participate in so many interactions is due in part to their subclass variation and further chemical modification. One such modification is glucosylation, where a glucose molecule is added to the flavonoid substrate. The enzymes that catalyze these reactions are known as glucosyltransferases. Citrus paradisi contains a glucosyltransferase that is specific to the 3-O position of flavonols. To further understand the reactions it catalyzes, Cp3-O-GT structure was modeled against an anthocyanidin/flavonol 3 GT found in Vitis vinifera to identify candidate amino acids for mutations. Mutants were then created using site-directed mutagenesis, and one mutant, D344P, was constructed by an aspartate being replaced with a proline based off of the sequence comparison of the original enzymes. Biochemically characterizing the mutant D344P protein will determine whether the mutation has an effect on the substrate specificity of Cp3-O-GT. An initial quickscreening assay using radioactive UDP-glucose as a sugar donor suggested there may have been expansion of substrate acceptance. Confirming time course assays did not support this. Additionally, results of these assays show that D344P protein has decreased activity with flavonols as compared to wild type Cp3-O-GT. with no expansion of substrate specificity. Models suggest that a change in protein conformation has resulted in decreased activity.

Flavonoids are a class of plant metabolites with C6-C3-C6 structure responsible for many biological functions, including coloration and defense. Citrus paradisi, grapefruit, contains a wide variety of flavonoids which are grouped by the extent of modification, examples of which are flavonols, flavones, and flavanones. A major modification is the addition of glucose by glucosyltransferases (GTs) to stabilize the structure and provide ease of transport. This process can be highly substrate and regiospecific. With Cp3OGT, glucose is added at the 3-hydroxy position. This 3GT only accepts flavonols as its substrate; however, a Vitis vinifera (grape) 3-GT can accept both flavonols and anthocyanidins. Homology modeling using the crystallized structure of the V. vinifera GT predicted sites of amino acids that could influence substrate binding site. The 382 position was of particular interest with arginine in C. paradisi and tryptophan in V. vinifera. This change is hypothesized to cause a shift in substrate specificity of the Cp3OGT to accept anthocyanidins as well as flavonols. Site-directed mutagenesis was performed to form the R382W mutant Cp3OGT and transformed into yeast for expression. Western blot determined the optimal protein induction period for the cells, after which the cells were broken to extract the recombinant mutant protein. Purification of the R382W 3GT allowed for enzyme analysis to be performed by measuring the incorporation of radioactive glucose into the reaction product. HPLC will be used to identify reaction products. An enzyme kinetics study will show the extent of any biochemical change in function as a result of this mutation; results will then be incorporated into a refined protein model.

Flavonoids are secondary metabolites that are important in plant defense, protection, and human health. Most naturally-occurring flavonoids are found in glucosylated form. Glucosyltransferases (GTs) are enzymes that catalyze the transfer of glucose from a high energy sugar donor to an acceptor molecule. At this time, it is not possible to accurately predict putative GT activity from sequence alone; biochemical characterization is critical. A flavonol-specific 3-O-GT enzyme has been identified and cloned from the leaf tissues of grapefruit. The enzyme shows rigid substrate specificity as well as regiospecificity. Several F3GT's characterized from other plants also had the ability to glucosylate anthocyanidins, however the grapefruit F3GT did not. This research is designed to test the hypothesis that specific amino acid residues impart the substrate specificity and regiospecificity of the grapefruit enzyme. Site-directed mutagenesis was performed on three potentially key amino acid residues within the grapefruit F3-GT that were identified through homology modelling. Enzyme activity of the mutant F3-GT proteins will be tested with flavonols for a possible change in glucosylation pattern. Other flavonoid classes will also be tested with the mutant F3-GT enzyme to test for change in substrate specificity. The result from this study will add to our knowledge of GTs.

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.