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-Oglucosyltransferase (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-GT- P145T 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.

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. The enzymes that lead to glucoside formation are known as glucosyltransferases (GTs), and characteristically accomplish this task by transferring a UDP-activated glucose to a corresponding acceptor molecule. 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 and limonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications in these species. The research focus of our lab is to establish the function of putative secondary product glucosyltransferase clones identified from Citrus paradisi. In the present study, we report on the activity and biochemical characterization of two clones, PGT 7 (Flavonol-3-O-GT) and PGT8 (Limonoid GT) which were expressed in Pichia pastoris.

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.

Plants produce a vast array of secondary metabolites. The phenolic compounds flavonoids are 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, reactions catalyzed by glucosyltransferases. Citrus paradise 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 modelled 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 regio and/or steriospecificity of Cp3-OGT. An initial screening assay has been performed using radioactive UDP-glucose as a sugar donor. Early results indicated that the mutant D344P has particular affinity for flavonols and for diosmetin, a flavone. Kinetic assays are being performed to confirm these results. Studies of time course, enzyme concentration, HPLC product analysis, pH optimum and reaction kinetics will be performed to further complete D344P protein characterization.

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.

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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.