Gene duplication and functional diversification is a central driving force in the evolution of plant biochemical diversity. However, the latter process is not well understood. Here the diversification of the plant shikimate/quinate dehydrogenase (S/QDH) gene family was investigated in order to shed light on how duplicate genes functionally diversify. The shikimate pathway is the major biosynthetic route towards the aromatic amino acids, linking vital protein biosynthesis with the production of aromatic secondary metabolites. Dehydroquinate dehydratase/shikimate dehydrogenase (SDH) encodes the central enzyme of this pathway, catalyzing the production of shikimate. Quinate is a secondary metabolite synthesized using the same precursors as shikimate by quinate dehydrogenase (QDH). Gene duplication prior to the gymnosperm / angiosperm split generated two distinct clades in seed plants separating SDH and QDH functions whereas non-seed plants have a single copy SDH. In vitro biochemical characterization of a reconstructed ancestral enzyme was performed alongside extant members separated prior to duplication (from a lycopod, a bryophyte, and a chlorophyte) and afterwards (from a gymnosperm and an angiosperm). This revealed that novel quinate biosynthetic activity was gained in seed plants, providing evidence for the diversification of gene function via neofunctionalization. However, the ability to use both NAD(H) and NADP(H) seems to have developed in both SDH and QDH clade members of angiosperms. Finally, a method is described for analysing quinate and its derivative, chlorogenic acid in transgenic Arabidopsis. / Graduate / 2021-05-11

No description available.

The shikimate pathway connects primary metabolism with the biosynthesis of the three aromatic amino acids (phenylalanine, tyrosine and tryptophan), which are essential protein building blocks. This pathway also provides precursors for a wide array of plant secondary metabolites with adaptive functions in plant adaptation and defense. The third and fourth steps of the shikimate pathway (the conversion of shikimate from 3-dehydroquinate via 3-dehydroshikimate) are catalyzed by a bi-functional enzyme called 3-dehydroquinate dehydratase/shikimate dehydrogenase (DQD/SDH). DQD/SDHs have been biochemically characterized in a few plant species including Arabidopsis thaliana, Solanum lycopersicum and Nicotiana tabacum. The embryo-lethal phenotype of Arabidopsis null mutants lacking DQD/SDH highlights a critical role of shikimate in primary metabolism. Quinate shares high structural similarity with shikimate and is an important secondary metabolite present in many plant species. Quinate and its derivatives (e.g. chlorogenic acid) serve important functions in plant defense due to their astringent (i.e. bitterness) and antimicrobial properties. Quinate can be derived from 3-dehydroquinate, and this reaction is catalyzed by quinate dehydrogense (QDH), the reaction mechanism of which resembles that of SDH. With a functional genomics approach, I demonstrated that two of the five poplar putative DQD/SDHs (Poptr1 and Poptr5, poplar DQD/SDH1 and 2) have exclusive specificity for shikimate, while the other three (Poptr2 to Poptr4, poplar QDH1 to 3) are involved in quinate biosynthesis. Phylogenetic reconstruction of the DQD/SDH/QDH superfamily has identified two distinct clades in seed plants that may act preferentially on either shikimate or quinate, whereas lineages that have diverged prior to the angiosperm/gymnosperm split, only have a single copy DQD/SDH. An evolutionary analysis was carried out, and the sequence of the immediate pre-duplication ancestral DQD/SDH (>300MYA) was estimated and reconstructed. Protein structure modelling and in vitro biochemical characterization of the ancestral recombinant protein was performed along with some extant members of this family (pre-duplication representatives: Rhodopirellula baltica (Rhoba), Chlamydomonas reinhardtii (Chlre), Physcomitrella patens (Phypa) and Selaginella moellendorffii (Selmo); post-duplication species: Pinus taeda (Pinta1 & Pinta2) and Populus trichocarpa (Poptr1 & Poptr3). Together, the results indicate that quinate biosynthetic activity was gained prior to duplication and remained low until it became beneficial and favored by selection. The optimization of quinate biosynthetic activity was at the expense of losing some primary shikimate biosynthetic function creating a pleiotropic conflict. This was then resolved by gene duplication and further specialization leading to genes encoding specialized enzymes (either SDH or QDH). Diversification of the DQD/SDH/QDH superfamily likely occurred through sub-functionalization via a mechanism described as “Escape from Adaptive Conflict.” / Graduate / 0307 / guojia@uvic.ca

The shikimate pathway links carbohydrate metabolism to biosynthesis of the aromatic amino acids in plants, fungi, bacteria and apicomplexan parasites. The pathway has seven enzymatic steps which convert erythrose-4-phosphate and phosphoenolpyruvate to chorismate, the precursor of tyrosine, tryptophan and phenylalanine. Due to the absence of the pathway in mammalian species, the enzymes are attractive targets for herbicides and antimicrobials. Shikimate dehydrogenase (SDH) catalyses the fourth step, the NADP-dependent reversible reduction of 3-dehydroshikimate to shikimate. Five SDH homologs – AroE, Ael1, YdiB, RifI and SdhL – have been identified through kinetic analysis and phylogenetic studies in the bacterium Pseudomonas putida. SDH homolog gene knockouts (KO) were used to characterize their functions. The AroE KO and Ael1 KO were successfully constructed via gene SOEing of the SDH homolog with a gentamycin antibiotic cassette and homologous recombination via electroporation into WT P. putida KT2440. Preliminary characterization tested KO growth, auxotroph recovery and fluorescent activity.

The shikimate pathway links carbohydrate metabolism to biosynthesis of the aromatic amino acids in plants, fungi, bacteria and apicomplexan parasites. The pathway has seven enzymatic steps which convert erythrose-4-phosphate and phosphoenolpyruvate to chorismate, the precursor of tyrosine, tryptophan and phenylalanine. Due to the absence of the pathway in mammalian species, the enzymes are attractive targets for herbicides and antimicrobials. Shikimate dehydrogenase (SDH) catalyses the fourth step, the NADP-dependent reversible reduction of 3-dehydroshikimate to shikimate. Five SDH homologs – AroE, Ael1, YdiB, RifI and SdhL – have been identified through kinetic analysis and phylogenetic studies in the bacterium Pseudomonas putida. SDH homolog gene knockouts (KO) were used to characterize their functions. The AroE KO and Ael1 KO were successfully constructed via gene SOEing of the SDH homolog with a gentamycin antibiotic cassette and homologous recombination via electroporation into WT P. putida KT2440. Preliminary characterization tested KO growth, auxotroph recovery and fluorescent activity.

No description available.

In field efficacy trials most glyphosate/surfactant combinations tested control barnyardgrass as well as preormulated glyphosate products 21 days after treatment (DAT). Preormulated glyphosate products controlled barnyardgrass quicker than combination treatments, most likely due to improved glyphosate formulations with improved surfactant systems. In greenhouse trials, preormulated glyphosate products exhibited greater fresh weight reductions across all species tested, including barnyardgrass, broadleaf signalgrass, hemp sesbania, johnsongrass, large crabgrass, pitted morningglory, prickly sida, sicklepod, yellow foxtail and Palmer amaranth. Nonionic tallow amine treatments exhibited variable control among species. A shikimate analysis was developed using non-glyphosate-tolerant soybean to estimate efficacy of surfactants; data were then correlated to the visual efficacy data on barnyardgrass in the field. However, there was not sufficient variability in barnyardgrass control to use the shikimate analysis as a predictor.

The shikimate pathway links carbohydrate metabolism to the biosynthesis of the aromatic amino acids and an enormous variety of aromatic compounds with essential functions in all kingdoms of life. Aromatic compounds derived from the plant shikimate pathway have substantial biotechnological value and many are essential to the diet of metazoans whose genomes do not encode shikimate pathway enzymes. Despite its importance to the physiology of plants and human health the regulatory mechanisms of the plant shikimate pathway are not well understood. Shikimate kinase (SK) genes encode an intermediate step in the shikimate pathway and were previously implicated in regulation of the plant shikimate pathway. The distribution of SK genes in higher plants was resolved using phylogenetic and biochemical methods. The two SK isoforms of Arabidopsis thaliana, AtSK1 and AtSK2, were functionally characterized. AtSK1 expression is induced by heat stress and the recombinant enzyme was shown to form a homodimer which is important for maintaining the stability and activity of the enzyme at elevated temperatures. The crystal structure of AtSK2, the first reported plant SK structure, identified structural features unique to plant SKs which may perform important regulatory functions. The resolution of bona fide SKs in higher plants led to the discovery of two novel neofunctionalized homologs - Shikimate Kinase-Like 1 (SKL1) and SKL2. These novel genes evolved from SK gene duplicates over 400 million years ago and are found in all major extant angiosperm lineages, suggesting they were important in the development of biological properties required by land plants. The description of albino and variegated skl1 mutants in Arabidopsis thaliana implicate the SKL1 gene product as an important regulator of chloroplast biogenesis. Functional assays were attempted to determine the biochemical function of SKL1 and recombinant constructs of the Arabidopsis thaliana SKL1 protein were crystallized towards structure determination. The results of this thesis further our understanding of the organization and regulation of the plant shikimate pathway. Furthermore, the discovery of SKL1 may yield important insights into chloroplast biogenesis and function. The evolution of the plant SK superfamily highlights the utility of SKs as scaffolds for functional innovation.

The shikimate pathway links carbohydrate metabolism to the biosynthesis of the aromatic amino acids and an enormous variety of aromatic compounds with essential functions in all kingdoms of life. Aromatic compounds derived from the plant shikimate pathway have substantial biotechnological value and many are essential to the diet of metazoans whose genomes do not encode shikimate pathway enzymes. Despite its importance to the physiology of plants and human health the regulatory mechanisms of the plant shikimate pathway are not well understood. Shikimate kinase (SK) genes encode an intermediate step in the shikimate pathway and were previously implicated in regulation of the plant shikimate pathway. The distribution of SK genes in higher plants was resolved using phylogenetic and biochemical methods. The two SK isoforms of Arabidopsis thaliana, AtSK1 and AtSK2, were functionally characterized. AtSK1 expression is induced by heat stress and the recombinant enzyme was shown to form a homodimer which is important for maintaining the stability and activity of the enzyme at elevated temperatures. The crystal structure of AtSK2, the first reported plant SK structure, identified structural features unique to plant SKs which may perform important regulatory functions. The resolution of bona fide SKs in higher plants led to the discovery of two novel neofunctionalized homologs - Shikimate Kinase-Like 1 (SKL1) and SKL2. These novel genes evolved from SK gene duplicates over 400 million years ago and are found in all major extant angiosperm lineages, suggesting they were important in the development of biological properties required by land plants. The description of albino and variegated skl1 mutants in Arabidopsis thaliana implicate the SKL1 gene product as an important regulator of chloroplast biogenesis. Functional assays were attempted to determine the biochemical function of SKL1 and recombinant constructs of the Arabidopsis thaliana SKL1 protein were crystallized towards structure determination. The results of this thesis further our understanding of the organization and regulation of the plant shikimate pathway. Furthermore, the discovery of SKL1 may yield important insights into chloroplast biogenesis and function. The evolution of the plant SK superfamily highlights the utility of SKs as scaffolds for functional innovation.

The emergence of antibiotic resistance to current treatments of bacterial infection represents a major challenge that needs to be addressed with the development of new generations of inhibitors. The enzyme 5-enolpyruvylshikimate 3- phosphate synthase (EPSPS) catalyses the sixth step in the shikimate biosynthetic pathway, which is essential for the synthesis of aromatic compounds such as the aromatic amino acids phenylalanine, tryptophan and tyrosine. It occurs in plants, bacteria and some parasites. Since the pathway is absent in mammals but essential for the pathogenicity of a number of organisms, EPSPS is considered a prospective target for new inhibiter design. A number of EPSPS inhibitors have been reported in the literature. What we are lacking is an understanding of the features that are important for binding EPSPS. We have synthesized compounds to probe the active site of the enzyme based on the knowledge of an enzyme-catalyzed intermediate with a high cationic character. This will include assembling bipartite/tripartite inhibitors to discover what interactions or structural motifs are important for binding. Once the features important for binding to EPSPS are understood, the possibility of elaborating them to create potent inhibitors of EPSPS will be investigated. In addition, the synthesis of two shikimate analogs [5-^(18)O] shikimic acid and 4-deoxyshikimic acid were completed for further experiments to probe the enzyme mechanism in detail, and for transition state structure by transition state analysis. Transition state analysis using kinetic isotopic effects (KIE) will elucidate the transition state structure of the enzyme-catalyzed EPSP reaction, and provide a detailed starting point for designing EPSPS inhibitors. / Thesis / Master of Science (MSc)