This research is focused on the study of the effect of mutating proline 145 to threonine on the substrate and regiospecificity of flavonol specific 3-O-glucosyltransferase (Cp3GT). While the mutant P145T enzyme did not glucosylate anthocyanidins, it did glucosylate flavanones and flavones in addition to retaining activity with flavonols. HPLC was used for product identification and showed mutant P145T glucosylated naringenin at the 7-OH position forming naringenin-7-O-glucoside and flavonols at the 3-OH position. Homology modeling and docking was done to predict the acceptor substrate recognition pattern and models were validated by experimental results. In other related work, a thrombin cleavage site was inserted into wild type Cp3GT and recombinant P145T enzyme between the enzyme and the C-myc tags in order to be able to cleave off tags. This provides the tool needed for future efforts to crystallize these proteins for structural determination.

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains xvii, 122 p. : ill. Includes abstract. Includes bibliographical references (p. 46-53).

Flavonoid and limonoid glycosides influence taste properties as well as marketability of Citrus fruit and products, particularly grapefruit. In this work, nine grapefruit putative natural product glucosyltransferases (PGTs) were resolved by either using degenerate primers against the semiconserved PSPG box motif, SMART-RACE RT-PCR, and primer walking to full-length coding regions; screening a directionally cloned young grapefruit leaf EST library; designing primers against sequences from other Citrus species; or identifying PGTs from Citrus contigs in the harvEST database. The PGT proteins associated with the identified full-length coding regions were recombinantly expressed in Escherichia coli and/or Pichia pastoris and then tested for activity with a suite of substrates including flavonoid, simple phenolic, coumarin, and/or limonoid compounds. A number of these compounds were eliminated from the predicted and/or potential substrate pool for the identified PGTs. Enzyme activity was detected in some instances with quercetin and catechol glucosyltransferase activities having been identified.

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.

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.

Petunia hybrida and Citrus paradisi have significantly different flavonoid accumulation patterns. Petunia sp. tend to accumulate flavonol glycosides and anthocyanins while Citrus paradisi is known for its accumulation of flavanone diglycosides. One possible point of regulation of flavanone metabolism is flavanone 3-hydroxylase (F3H) expression. To test whether this is a key factor in the different flavanone usage by Petunia hybrida and Citrus paradisi, F3H mRNA expression in seedlings of different developmental stages was measured using semi-quantitative RT-PCR. Primers were designed to conserved regions of F3H and used to amplify an approximately 350 bp segment for quantitation by PhosphorImaging. Primary leaves of 32 day old grapefruit seedlings and a grapefruit flower bud had the highest levels of F3H mRNA expression. Petunia seedlings had much lower levels of F3H mRNA expression relative to grapefruit. The highest expression in petunia was in primary leaves and roots of 65 day old seedlings. These results indicate that preferential use of naringenin for production of high levels of flavanone glycosides in young grapefruit leaves cannot be attributed to decreased F3H mRNA expression.

Flavonoids are secondary metabolites that have significant roles in plant defense and human nutrition. Glucosyltransferases (GTs) catalyze the transfer of sugars from high energy sugar donors to other substrates. Several different secondary product GTs exist in the tissues of grapefruit making it a model plant for studying their structure and function. The goal of this investigation was to determine the expression patterns of seven putative secondary product GTs during grapefruit growth and development by quantifying mRNA expression levels in the roots, stems, leaves, flowers, and mature fruit to establish whether the genes are expressed constitutively or if one or more could be expressed in a tissue specific manner and/or developmentally regulated. Six growth stages were defined from which RNA was extracted, and expression levels were quantified by standardized densitometry of gene-specific RT-PCR products. Results show that there were variable degrees of PGT expression in different tissues and at different developmental stages. These results add to the growing knowledge base of dynamics of expression and potential regulation of secondary metabolism in Citrus paradisi.

Glucosylation is a predominant flavonoid modification reaction affecting the solubility, stability, and subsequent bioavailability of these metabolites. Flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications in these species. In this work, a Citrus paradisi glucosyltransferase gene was identified, cloned, and introduced into the pET recombinant protein expression system utilizing primers designed against a predicted flavonoid glucosyltransferase gene (AY519364) from Citrus sinensis. The encoded C. paradisi protein is 51.2 kDa with a predicted pI of 6.27 and is 96% identical to the C. sinensis homologue. A number of compounds from various flavonoid subclasses were tested, and the enzyme glucosylated only the flavonol aglycones quercetin (Kmapp = 67 μ M; Vmax = 20.45 pKat/μg), kaempferol (Kmapp = 12 μ M; Vmax = 11.63 pKat/μg), and myricetin (Kmapp = 33 μ M; Vmax = 12.21 pKat/μg) but did not glucosylate the anthocyanidin, cyanidin. Glucosylation occurred at the 3 hydroxyl position as confirmed by HPLC and TLC analyses with certified reference compounds. The optimum pH was 7.5 with a pronounced buffer effect noted for reactions performed in Tris-HCl buffer. The enzyme was inhibited by Cu2+, Fe2+, and Zn2+ as well as UDP (Kiapp = 69.5 μ M), which is a product of the reaction. Treatment of the enzyme with a variety of amino acid modifying compounds suggests that cysteine, histidine, arginine, tryptophan, and tyrosine residues are important for activity. The thorough characterization of this C. paradisi flavonol 3-O-glucosyltransferase adds to the growing base of glucosyltransferase knowledge, and will be used to further investigate structure-function relationships.

Flavonoids are chemically modified by glucosylation, hydroxylation, methylation, etc. During glucosylation, the sugar moiety from UDP-sugar is transferred to aglycone flavonoid substrates by glucosyltransferases (GTs). Grapefruit contains 5 different glucosyltransferases that demonstrate differences in not only substrate but also position specificity. Previous research obtained 3 putative 5’ grapefruit GT clones using SMART RACE RT-PCR with a degenerate gene specific primer based on a highly conserved sequence area in the Plant Secondary Product Glucosyltransferase box. The objective of this research was to use clone specific primers to obtain 3’ clones of the 3 previously mentioned 5’ clones as well as verify putative GT candidacy based on sequence data. Two of the 3 putative GT candidates were designated non-GTs following 3’end sequencing. During pursuit of sequence for the remaining 5’ clone, 1 full-length clone and 1 partial putative GT clone were obtained. To verify GT status, the clones must undergo expression/biochemical characterization.