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.

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.

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.

Flavonoids are a class of plant metabolites with a C6-C3-C6 structure. They are responsible for a large range of biological functions including UV protection, pigmentation, and anti-microbial properties. Citrus paradisi, the grapefruit, contains a wide variety of flavonoids, including the target flavonols which are characterized by a hydroxyl group at the C3 position. A glucose molecule is added to flavonols by 3-Oglucosyltransferases (3-O-GTs). C. paradisi F3-O-GT only glucosylates flavonols; however, Vitis vinifera (grape) 3-O-GT can accept both flavonols and anthocyanidins. The two enzymes have some identity with one another but sequence alignment pinpointed several areas of non-homology. Homology modeling using the crystallized structure of the V. vinifera 3-GT revealed sites within the non-homologous areas that could influence the binding site most directly. The 382 site was of particular interest with arginine in C. paradisi changed to tryptophan in V. vinifera, a much bulkier and non-charged amino acid. Site-directed mutagenis was performed to form the R382W mutant line and transformed into yeast for expression after induction with methanol. Western blot was used to determine the optimal protein induction time, after which the cells were harvested and broken to extract the proteins. Isolation and purification of the protein in question allows for enzyme analysis. This is performed by measuring incorporation of radioactive glucose onto various substrates from each flavonoid class. High counts indicate that the enzyme is active upon the substrate while low counts indicate little to no activity. Characterization will also be performed by varying reaction conditions. Thus, the optimal pH, temperature, substrate quantity, enzyme quantity, and reaction duration can be determined for this specific mutant. These experiments will determine if the R382W mutation has a significant impact on the substrate specificity or reaction conditions for the enzyme. A change in activity to include other classes of flavonoids besides flavonols indicates that the mutation site has a direct impact on the conformation of the binding site. Failure of the mutation to change substrate specificity still provides valuable information for the structure and function of the enzyme. This has implications for engineering enzymes to perform specific functions.