Citrus fruits are widely consumed and can offer various health benefits. One enzyme found in grapefruits, Citrus paradisi flavonol specific 3-O-glucosyltransferase (CP3GT), catalyzes the addition of glucose to one specific flavonoid class and at only one site. These flavonoids are plant secondary metabolites that can be used in a variety of plant functions including signaling and protection. The only class of flavonoids that CP3GT glucosylates is flavonols, and this specificity is of interest to study for potential benefits in biotechnology and enzyme modeling. In order to study this enzyme and its structure, a variety of mutants were created using site-directed mutagenesis. One mutant, P297F, exhibited a loss of function. This mutant was previously studied by inserting a thrombin cleavage site, extracting the plasmid expressing the mutation and sequencing it. The gene sequence was previously verified to be in frame and contain the needed thrombin cleavage site to remove tags used for protein purification and identification. The plasmid was then linearized, and transformed into yeast. After this, conditions for protein expression were tested over a 72-hour period. The protein was found to have optimal expression at 50 hours with a constant temperature of 28 °C and methanol concentration of 0.5 %. However, numerous protein expression experiments indicated very low protein expression. For this reason, the P297F gene was amplified through colony PCR, extracted and sent for sequencing to verify the transformation of the gene into yeast and identify possible reasons for low protein production. Analysis of this sequencing data showed a single nucleotide addition early in the tag sequence causing a frameshift after this location. Reanalysis of the previous plasmid sequencing data showed this same mutation, indicating improper conclusions were drawn. Efforts should be made to identify a plasmid without the mutation or correct the frameshift mutation so that the tag sequence produces the correct amino acids.

Flavonoids are plant secondary metabolites that are integral to our lives. Grapefruits are well-known for production of unique glucosylated products and the enzymes responsible are UDP-glucose:glucosyltransferases (GTs). The objective of this research was to obtain full-length clones of putative grapefruit GTs, express them, and characterize them. Previously, gene specific primers (from conserved PSPG box) and clone specific primers (from partial 5' clones) were designed, and a compiled sequence attained using SMART RACE RT-PCR. A full-length clone was obtained using primers designed from the extreme ends of the compiled sequence. The full-length clone was inserted into expression vector (pET32a) and transformed into expression host BL21(DE3)RIL. Expressed protein was tested for GT activity using different flavonoid aglycones and UDP-14C-glucose as glucose donor. Results indicated that the expressed protein was probably not a flavonoid GT. A directionally cloned grapefruit leaf cDNA library is undergoing EST mining to identify additional GT candidates.

Flavonoids are plant secondary metabolites that play diverse roles in plants and human health. These compounds in most part exist in the glucosylated form. Grapefruit accumulates high levels of glucosylated flavonoids. Plant secondary product glucosyltransferases (GTs) catalyze the glucosylation reaction, but due to low homology at both the nucleotide and amino acid sequence level of different GTs, it is not possible to ascribe function based on sequence only. The hypotheses that PGT clones 9 and 11 are plant secondary product GTs and are biochemically regulated were tested. PGT 9 has been cloned into Pichia pastoris using the pPICZA and pPICZAα vectors, expressed, enriched, and screened for GT activity with a variety of phenolic substrates. Initial screens show catechol, gentisic acid, vanillin, and p-hydroxylphenylacetic acid as potential substrates for the PGT 9 protein. PGT 11 has been successfully cloned into pPICZA for transformation into yeast, expression, and subsequent characterization.

The grapefruit plant, Citrus paradisi, tends to accumulate high levels of flavonoid glycosides such as flavanones and flavones. Flavonoids have a vast array of important functions in plants and also in humans. Glucosyltransferases (GTs) are enzymes responsible for glucosylation reactions. In our pursuit to study the structure and function of flavonoid GTs, we have used molecular approaches to identify, clone, express, and functionally characterize the enzymes. This research was designed to test the hypothesis that PGT3 is a flavonoid glucosyltransferase and is subject to biochemical regulation. PGT3 has been tested for GT activity with compounds representing subclasses of flavonoids as well as some simple phenolics. Results indicate GT activity with 6 substrates, p-hydroxybenzoic acid, vanillin, vanillic acid, p-hydroxyphenylpyruvate, gentisic acid, and catechol. A second project designed to clone putative PGT8 into the Pichia expression system has been completed.

Glucosyltransferases catalyze the transfer of glucose molecules from an active donor to acceptor molecules and are involved in many plant processes. SIP68, a tobacco glucosyltransferase protein, is a SABP2-interacting protein. It was identified in a yeast two-hybrid screen using SABP2 as bait and tobacco proteins as prey. SABP2, converts methyl salicylate to salicylic acid (SA) as a part of the signal transduction pathways in SA-mediated defense signaling. Subcellular localization is a crucial aspect of protein functional analysis to assess its biological function. The recombinant SIP68 tagged with eGFP was expressed transiently in Nicotiana benthamiana and observed under confocal microscopy. Fluorescent signals were observed in the epidermal cells. Subcellular fractionation of the tobacco leaves transiently expressing SIP68-+eGFP confirmed that SIP68 is localized in the cytosol. To study the role of SIP68 in plant stress signaling, transgenic lines with altered SIP68 expression were generated using RNAi and CRISPR Cas9 and analyzed.

Analysis of SIP68: A UDP Glucosyltransferase for Its Role in Plant Growth and Immunity UDP-glucosyltransferases (GTs) are a group of enzymes that play a crucial role in plant metabolism by transferring glucosyl groups from UDP-glucose to various acceptor molecules. SIP68 is a UDP-glucosyltransferase enzyme that has been identified to interact with SABP2 in a yeast two-hybrid screen. Previous research conducted in our lab has demonstrated that SIP68 is involved in salicylic acid (SA)-mediated defense signaling in tobacco plants. In the current study, we aimed to investigate the potential role of SIP68 in plant development and immune response. Our analysis of SIP68 revealed that this UDP-glucosyltransferase has a gene family, and its gene and protein sequence, molecular attributes, gene structure, and localization in the chromosome, exon-intron distribution, cis-regulatory elements in the promoter region, homology modeling of protein, domain architecture, motif analysis, phylogenetic tree, and protein-protein interaction were analyzed to better understand its potential function in plant metabolism. Our in-silico analysis predicted that SIP68 may play a role in the cytokinin-mediated metabolic pathway, which could affect plant growth and cell proliferation. Specifically, our analysis suggested that SIP68 might transfer glucosyl groups to various acceptor molecules involved in the cytokinin-mediated metabolic pathway. This suggests that SIP68 may play a role in regulating plant growth and development by affecting the cytokinin pathway. To investigate the potential role of SIP68 in plant development, we generated SIP68-deficient transgenic tobacco plants by silencing the SIP68 protein. The observed phenotype of these plants was compared to that of wild-type plants. We found that root, shoot, leaf width, and overall biomass development were all affected in SIP68-deficient plants. This suggests that SIP68 plays a crucial role in regulating various aspects of plant growth and development. This agrees with our previous finding that SIP68 is involved in SA-mediated defense signaling in tobacco plants. Our analysis of protein-protein interactions revealed that SIP68 interacts with various classes of flavanols in-vitro. This interaction provides a starting point for investigating potential targets of SIP68 in tobacco plants. However, the specific in-planta substrate(s) of SIP68 has not yet been identified. Therefore, further investigation is needed to determine the intracellular targets of SIP68 and its specific role in plant metabolism. In conclusion, our study provides insights into the potential role of SIP68 in plant development and immune response. Our findings suggest that SIP68 plays a crucial role in regulating various aspects of plant growth and development. Furthermore, our in-silico analysis predicts that SIP68 may play a role in the cytokinin-mediated metabolic pathway, which could affect plant growth and cell proliferation. Future investigation is needed to determine the intracellular targets of SIP68 and its specific role in plant metabolism. Overall, this study highlights the importance of UDP-glucosyltransferase enzymes (SIP68) in plant development and immune response.

Plant secondary metabolites are essential for normal growth and development in plants ultimately affecting crop yield. They play roles ranging from appearance of the plants to defending against pathogen attack and herbivory. They have been used by humans for medicinal and recreational purposes amongst others. Glycosyltransferases catalyze the transfer of sugars from donor substrates to acceptors. Glucosyltransferases are a specific type of glycosyltransferases known to transfer glucose molecules from a glucose donor to a glucose acceptor (aglycone) producing the corresponding glucose secondary metabolite or glycone, in this case glucosides. It was hypothesized that SBIP68, a tobacco putative glucosyltransferase-like protein glucosylated salicylic acid. Salicylic acid is an essential plant defense secondary metabolite. SBIP68 was cloned and heterologously expressed in both prokaryotic and eukaryotic systems. Results from activity screening suggest that SBIP68 is a UDP-glucose flavonoid glucosyltransferase with broad substrate specificity. Further studies are required to fully characterize SBIP68.

Flavonoids are a broad class of low molecular weight, secondary plant phenolics characterized by the fl avan nucleus. Widely distributed in plants, food and traditional herbal medicines, more than 6000 fl avonoids have been identifi ed up to date. They are present mainly as glycosides whose phenolic hydrogen or hydrogens are substituted to sugar moiety. An increasing number of fl avonoids have attracted much attention in relation to their biological activities, including anti-viral, anti-infl ammatory, anti-bacterial, and vasodilatory activities. Present work is to understand the structure and function of a fl avonol specifi c glucosyltransferase from Citrus paradisi. The study is one of the many steps towards custom designing of the protein. We employed homology modeling, site-directed mutagenesis and yeast expression system to generate mutants of glucosyltransferase and study their substrate specifi city, regiospecifi city and kinetic properties.

This thesis investigates two aspects of phytoalexin metabolism by the phytopathogenic fungus <i>Sclerotinia sclerotiorum</i> (Lib) de Bary: (i) determination of detoxification pathways of structurally different molecules; (ii) design and synthesis of potential inhibitors of enzyme(s) involved in detoxification steps.<p>First, the transformations of important cruciferous phytoalexins by the economically important stem rot fungus, <i>S. sclerotiorum</i>, were investigated. During these studies a number of new metabolic products were isolated, their chemical structures were determined using spectroscopic techniques, and further confirmed by synthesis. The metabolic products did not show detectable antifungal activity against <i>S. sclerotiorum </i> which indicated that these metabolic transformations were detoxification processes. Overall, the results of these transformations suggested that <i>S. sclerotiorum</i> produces various enzymes that can detoxify cruciferous phytoalexins via different pathways. While the detoxifications of strongly and moderately antifungal phytoalexins such as brassilexin, sinalexin, and 1-methoxybrassinin were fast and led to glucosylated products, the transformations of the weakly antifungal phytoalexins brassicanal A, spirobrassinin and 1-methoxyspirobrassinin were very slow and yielded non-glucosylated compounds.<p>Next, the design of potentially selective inhibitors of the brassinin detoxification enzyme, BGT, was sought. Two sets of potential inhibitors of BGT were designed: (i) a group was based on the structure of brassinin, where the indole ring of brassinin was replaced with benzofuran, thianaphthene, 7-azaindole and pyrazolo[1,5-a]pyridine and/or the position of side chain was changed from C-3 to C-2; and (ii) another group based on the structure of camalexin where the thiazole ring of camalexin was replaced with a phenyl group. The syntheses and chemical characterization of these potential detoxification inhibitors, along with their antifungal activity, as well as screening using fungal cultures and cell-free extracts of <i>S. sclerotiorum</i>, were examined. The results of these screening indicated that 3-phenylindoles, 3-phenylbenzofuran, 5-fluorocamalexin, methyl (indol-2-yl)methyl-dithiocarbamate, methyl (benzofuran-3-yl)methyldithiocarbamate and methyl (benzo-furan-2-yl)methyldithiocarbamate could slow down the rate of detoxification of brassinin in fungal cultures and also in cell-free extracts of <i>S. sclerotiorum</i>. Among the designed compounds, 3-phenylindole appeared to be the best inhibitor both in fungal cultures and in cell-free extracts. Metabolism studies of all the designed compounds using fungal cultures of <i>S. sclerotiorum</i> indicated that they were metabolized by <i>S. sclerotiorum</i> to glucosyl derivatives, although at much slower rates.<p>It is concluded that some inhibitors that can slow down the rate of metabolism of brassinin could be good leading structures to design more active inhibitors of BGT.

This thesis investigates two aspects of phytoalexin metabolism by the phytopathogenic fungus <i>Sclerotinia sclerotiorum</i> (Lib) de Bary: (i) determination of detoxification pathways of structurally different molecules; (ii) design and synthesis of potential inhibitors of enzyme(s) involved in detoxification steps.<p>First, the transformations of important cruciferous phytoalexins by the economically important stem rot fungus, <i>S. sclerotiorum</i>, were investigated. During these studies a number of new metabolic products were isolated, their chemical structures were determined using spectroscopic techniques, and further confirmed by synthesis. The metabolic products did not show detectable antifungal activity against <i>S. sclerotiorum </i> which indicated that these metabolic transformations were detoxification processes. Overall, the results of these transformations suggested that <i>S. sclerotiorum</i> produces various enzymes that can detoxify cruciferous phytoalexins via different pathways. While the detoxifications of strongly and moderately antifungal phytoalexins such as brassilexin, sinalexin, and 1-methoxybrassinin were fast and led to glucosylated products, the transformations of the weakly antifungal phytoalexins brassicanal A, spirobrassinin and 1-methoxyspirobrassinin were very slow and yielded non-glucosylated compounds.<p>Next, the design of potentially selective inhibitors of the brassinin detoxification enzyme, BGT, was sought. Two sets of potential inhibitors of BGT were designed: (i) a group was based on the structure of brassinin, where the indole ring of brassinin was replaced with benzofuran, thianaphthene, 7-azaindole and pyrazolo[1,5-a]pyridine and/or the position of side chain was changed from C-3 to C-2; and (ii) another group based on the structure of camalexin where the thiazole ring of camalexin was replaced with a phenyl group. The syntheses and chemical characterization of these potential detoxification inhibitors, along with their antifungal activity, as well as screening using fungal cultures and cell-free extracts of <i>S. sclerotiorum</i>, were examined. The results of these screening indicated that 3-phenylindoles, 3-phenylbenzofuran, 5-fluorocamalexin, methyl (indol-2-yl)methyl-dithiocarbamate, methyl (benzofuran-3-yl)methyldithiocarbamate and methyl (benzo-furan-2-yl)methyldithiocarbamate could slow down the rate of detoxification of brassinin in fungal cultures and also in cell-free extracts of <i>S. sclerotiorum</i>. Among the designed compounds, 3-phenylindole appeared to be the best inhibitor both in fungal cultures and in cell-free extracts. Metabolism studies of all the designed compounds using fungal cultures of <i>S. sclerotiorum</i> indicated that they were metabolized by <i>S. sclerotiorum</i> to glucosyl derivatives, although at much slower rates.<p>It is concluded that some inhibitors that can slow down the rate of metabolism of brassinin could be good leading structures to design more active inhibitors of BGT.