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.

Deuteration is the replacement of a hydrogen atom by deuterium atom in a molecule. The replacement begins at the most acidic hydrogen in the molecule. In ATP, the deshielded hydrogen is C8-H which is the first replaced during deuteration. During ATP deuteration some of the ATP is hydrolysed to ADP concurrently. Using kinetic analysis, it was confirmed that the ATP hydrolysis that occurs is 1st order in ATP concentration, while the hydrogen replacement is 2nd order. The ATP and its C8 deuterated analogue were tested against three enzymes shikimate kinase (SK), acetate kinase (AK) and glutamine synthetase (GS) to determine if a kinetic isotope effect (KIE) exists in these systems. With AK and GS, the KIED increased as the KIEH decreased, while with SK the KIED decreased as the KIEH increased as the concentration of the ATP or deuterated analogue increased. Deuteration of imidazole and purine compounds reduced the specific activity of AK or SK at low concentrations in an enzyme-catalysed reaction. From a library of imidazole-containing compounds that inhibited SK, three compounds were selected and their IC50 values were determined on the SK-catalysed reaction. These compounds show a differential potency and efficiency between their protonated and deuterated analogues when compared in a 1:1 mixture. Synthesized purines incorporating three different substituents at N-9 were tested against AK or SK for their ability to lower the specific activity of the enzymes used / Physics / M. Sc. (Physics)

Deuteration is the replacement of a hydrogen atom by deuterium atom in a molecule. The replacement begins at the most acidic hydrogen in the molecule. In ATP, the deshielded hydrogen is C8-H which is the first replaced during deuteration. During ATP deuteration some of the ATP is hydrolysed to ADP concurrently. Using kinetic analysis, it was confirmed that the ATP hydrolysis that occurs is 1st order in ATP concentration, while the hydrogen replacement is 2nd order. The ATP and its C8 deuterated analogue were tested against three enzymes shikimate kinase (SK), acetate kinase (AK) and glutamine synthetase (GS) to determine if a kinetic isotope effect (KIE) exists in these systems. With AK and GS, the KIED increased as the KIEH decreased, while with SK the KIED decreased as the KIEH increased as the concentration of the ATP or deuterated analogue increased. Deuteration of imidazole and purine compounds reduced the specific activity of AK or SK at low concentrations in an enzyme-catalysed reaction. From a library of imidazole-containing compounds that inhibited SK, three compounds were selected and their IC50 values were determined on the SK-catalysed reaction. These compounds show a differential potency and efficiency between their protonated and deuterated analogues when compared in a 1:1 mixture. Synthesized purines incorporating three different substituents at N-9 were tested against AK or SK for their ability to lower the specific activity of the enzymes used / Physics / M. Sc. (Physics)

Tuberculosis (TB) is an infectious and fatal disease that ranks as the second leading killer worldwide. It is caused by Mycobacterium tuberculosis (Mtb) which is an obligate intracellular parasite that colonizes the alveolar macrophages of the immune system. The major health concern associated with TB is its co-infection with HIV and the development of strains with multi-drug resistance. The elimination of TB has been hindered due to the lack of understanding of the survival strategies used by this pathogen. Thus, research towards discovering new effective antibacterial drugs is necessary and a group of Mtb kinase enzymes were targeted in this study because these enzymes are crucial for metabolism, pathogenesis and, hence, the survival of Mtb. Kinases are a group of structurally distinct and diverse proteins that catalyze the transfer of the phosphate group from high energy donor molecules such as ATP (or GTP) to a substrate. The phosphorylation of proteins modifies the activity of specific proteins which is subsequently used to control complex cellular processes within Mtb. The starting point of this research targeted eight specific Mtb kinases namely; Nucleoside diphosphokinase, Homoserine kinase, Acetate kinase, Glycerol kinase, Thiamine monophosphate kinase, Ribokinase, Aspartokinase and Shikimate kinase. The aim of this project was to subclone the gene sequences for these eight recombinant Mtb kinases and express them in Escherichia coli, to purify the proteins and determine their activity. In the effort to find new lead compounds, the final stage of this study focused on the basic screening of the TB kinases against an extract prepared from Pelargonium sidoides, a medicinal plant, to identify any inhibitory effects. Although this traditional medicinal plant has been broadly researched and extensively used to treat TB, there is still a lack of understanding of this plant’s scientific curative effect. Various molecular and biochemical methods were used to achieve the aims of this project. The putative gene sequence was obtained from the annotated genome of H37Rv, deposited at NCBI as NC_000962.2. The genes encoding the kinases were successfully PCR-amplified from genomic DNA, cloned into an expression vector in-frame with a C- or N-terminal 6-histidine-tag and expressed in E. coli BL21 (DE3). The purification of the protein was complex, but various different methods and techniques were explored to obtain sufficient amounts of protein. The functional characterization of the kinases involved an HPLC enzyme assay that showed that the recombinant kinases were active. These enzymes were then screened against the potential inhibitory compounds in P. sidoides using enzyme assays to generate dose-response curves. This allowed an effective comparison not only of the Mtb kinases’ activity under normal conditions but also the kinases’ activity in the presence of a potential inhibitor. Overall, the inhibition of the enzymes required the presence of higher concentrations of the P. sidoides extract. However, the SK enzyme results presented a significantly higher inhibition and the lowest IC50 value, in comparison to the other kinases, which makes this kinase an attractive potential drug target against TB. In summation, cloning and purification of SK was successful, resulting in a concentration of 2030 μg/ml of purified enzyme and its activity analysis demonstrated enzyme functionality. This activity was reduced to zero in the presence of 1 x 102 mg/ml dilution of P. sidoides plant extract. This research conducted has extended the quality of information available in this field of study. These interesting results, proposing and identifying SK as a suitable potential target can be a starting point to significantly contribute and progress in this field of research, with the eventual goal of developing a drug to combat this fatal disease. / Life Sciences / M. Sc. (Life Sciences)

Tuberculosis (TB) is an infectious and fatal disease that ranks as the second leading killer worldwide. It is caused by Mycobacterium tuberculosis (Mtb) which is an obligate intracellular parasite that colonizes the alveolar macrophages of the immune system. The major health concern associated with TB is its co-infection with HIV and the development of strains with multi-drug resistance. The elimination of TB has been hindered due to the lack of understanding of the survival strategies used by this pathogen. Thus, research towards discovering new effective antibacterial drugs is necessary and a group of Mtb kinase enzymes were targeted in this study because these enzymes are crucial for metabolism, pathogenesis and, hence, the survival of Mtb. Kinases are a group of structurally distinct and diverse proteins that catalyze the transfer of the phosphate group from high energy donor molecules such as ATP (or GTP) to a substrate. The phosphorylation of proteins modifies the activity of specific proteins which is subsequently used to control complex cellular processes within Mtb. The starting point of this research targeted eight specific Mtb kinases namely; Nucleoside diphosphokinase, Homoserine kinase, Acetate kinase, Glycerol kinase, Thiamine monophosphate kinase, Ribokinase, Aspartokinase and Shikimate kinase. The aim of this project was to subclone the gene sequences for these eight recombinant Mtb kinases and express them in Escherichia coli, to purify the proteins and determine their activity. In the effort to find new lead compounds, the final stage of this study focused on the basic screening of the TB kinases against an extract prepared from Pelargonium sidoides, a medicinal plant, to identify any inhibitory effects. Although this traditional medicinal plant has been broadly researched and extensively used to treat TB, there is still a lack of understanding of this plant’s scientific curative effect. Various molecular and biochemical methods were used to achieve the aims of this project. The putative gene sequence was obtained from the annotated genome of H37Rv, deposited at NCBI as NC_000962.2. The genes encoding the kinases were successfully PCR-amplified from genomic DNA, cloned into an expression vector in-frame with a C- or N-terminal 6-histidine-tag and expressed in E. coli BL21 (DE3). The purification of the protein was complex, but various different methods and techniques were explored to obtain sufficient amounts of protein. The functional characterization of the kinases involved an HPLC enzyme assay that showed that the recombinant kinases were active. These enzymes were then screened against the potential inhibitory compounds in P. sidoides using enzyme assays to generate dose-response curves. This allowed an effective comparison not only of the Mtb kinases’ activity under normal conditions but also the kinases’ activity in the presence of a potential inhibitor. Overall, the inhibition of the enzymes required the presence of higher concentrations of the P. sidoides extract. However, the SK enzyme results presented a significantly higher inhibition and the lowest IC50 value, in comparison to the other kinases, which makes this kinase an attractive potential drug target against TB. In summation, cloning and purification of SK was successful, resulting in a concentration of 2030 μg/ml of purified enzyme and its activity analysis demonstrated enzyme functionality. This activity was reduced to zero in the presence of 1 x 102 mg/ml dilution of P. sidoides plant extract. This research conducted has extended the quality of information available in this field of study. These interesting results, proposing and identifying SK as a suitable potential target can be a starting point to significantly contribute and progress in this field of research, with the eventual goal of developing a drug to combat this fatal disease. / Life Sciences / M. Sc. (Life Sciences)