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.

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.

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Flavonoids are a class of secondary metabolites, the majority of which are present in glucosylated form. Glucosyltransferases are the enzymes that mediate glucosylation by transferring glucose from a high energy sugar donor to the acceptor substrates. Our study focuses on the structural and functional analysis of a flavonol-specific 3-O-glucosyltransferase (Cp-3-O-GT) clone from Citrus paradisi that has been characterized previously in our lab. 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-OGT. 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 flavonols 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 7-OH position forming prunin. Product identification with other substrates is in progress. Results are being used to revisit and refine the structure model. Structural and functional analysis of flavonoid GTs may contribute to custom design of GTs for the synthesis of novel glucosides by changing glucosylation patterns.

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 a 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 diosometin, 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.