<p>  </p> <p>With the increasing concern of climate change, engineering strategies to capture and fix carbon dioxide to produce valuable chemicals is a promising proposition. Metabolic engineering efforts have recently been focused on using cyanobacteria as hosts for the production biochemicals due to their ability to utilize carbon dioxide and sunlight as the sole carbon and energy sources, respectively. Unlike fermentation which uses plant derived sugars, cyanobacterial biochemical production does not compete for arable land that can be utilized for food production. Aromatic amino acids such as L-phenylalanine (Phe) and L-tryptophan (Trp) are essential amino acids since they cannot be synthesized by animals and thus are needed as supplements. They are valuable as animal feed supplements in the agricultural industry and find wide applications in the food, cosmetic and pharmaceutical industries as precursors. However, investigation of cyanobacteria for production of aromatic amino acids such as Phe and Trp is limited. This dissertation studies (<em>i</em>) combining random mutagenesis and metabolic engineering techniques for Trp and Phe production in <em>Synechocystis </em>sp. PCC 6803, (<em>ii</em>) development of a fast-growing cyanobacteria strain <em>Synechococcus elongatus</em> PCC 11801 for Phe production and (<em>iii</em>) investigating the effect of creation of Phe sink on photosynthetic efficiency under different light intensities.</p> <p>Aromatic amino acid biosynthesis is tightly regulated by feedback inhibition in cyanobacteria. To enable overproduction of Trp in <em>Synechocystis</em> sp PCC 6803, we utilized chemical mutagenesis coupled with analog selection followed by genome sequencing to identify single nucleotide polymorphisms (SNPs) responsible for the Trp overproduction phenotypes. Interestingly, overproducers had mutations in the competing Phe biosynthetic pathway gene chorismate mutase (CM) which resulted in a lower enzyme activity and redirection of flux to Trp. We subsequently overexpressed genes encoding feedback insensitive enzymes in our randomly engineered Trp overproducing strain. The best strain isolated was able to accumulate 212±23 mg/L Trp in 10 days under 3% (vol/vol) CO2. We demonstrate that combining random mutagenesis and metabolic engineering is superior to either approach alone.</p> <p>Initial efforts in engineering cyanobacteria have resulted in low titers and productivities due to slow growth. Recently a fast-growing cyanobacterial strain <em>Synechococcus elongatus</em> PCC 11801 was discovered with growth rates comparable to yeast. Due to the lack of well characterized synthetic biology tools available for metabolic engineering of this strain, we use two rounds of ultraviolet (UV) mutagenesis and analog selection to develop Phe overproducing strains. The best strain obtained using this strategy can produce 1.2 ± 0.1 g/L of Phe in 3 days under 3% (vol/vol) CO2. This is the highest titer and productivity for Phe production currently reported by cyanobacteria highlighting the promise of engineering fast-growing strains for biochemical production.</p> <p>Interestingly, Phe overproduction does not compete with growth but happens by fixing carbon at a higher rate. It is thought that the introduction of this carbon and energy sink relieves “sink limitation” by improving light use. However, neither the molecular mechanism nor the effect of light on enhancement in carbon fixation by introduction of an additional sink are known. Therefore, we investigated the effect of light intensity on photosynthetic efficiency, linear and cyclic electron flow in the strain containing the Phe sink. Our results indicate that under excess light, introduction of the Phe sink improves carbon fixation by improving photosynthetic efficiency and substantially reducing the cyclic electron flow around photosystem I (PSI). Taken together, our results show the previously untapped potential of cyanobacteria to improve carbon fixation by the unintuitive strategy of introducing a native carbon product sink and highlight the importance of the light environment on its performance.</p> <p>Although further improvements in titer, productivity, and scale up will be necessary for cyanobacteria to compete economically at the industrial scale, this dissertation adds to the scientific knowledge and techniques for further metabolic engineering efforts.</p>

Salicylic acid is a plant hormone that accumulates with plant-pathogen interaction. This accumulation corresponds to the plant being resistant to infection and without it the plant is susceptible. In this study, primers of genes involved in the normal synthesis of SA were used in RT-PCR to compare gene expression levels in susceptible and resistant plants challenged with tobacco mosaic virus. Because SA synthesis shares chorismate as a common substrate with the synthesis of aromatic amino acids, HPLC was used to determine whether the increase in SA could be attributed to a decrease in amino acid levels. The results suggest that genes of the shikimate pathway are up-regulated in both plant lines but much more quickly in the resistant plant, making differential gene expression a possible cause of SA accumulation. Additionally, results showed a more pronounced decrease in amino acid levels in resistant plants compared to susceptible plants.

Apesar da incidência per capita da tuberculose (TB) ter se mantido estável em 2005, o número de novos casos que surgem a cada ano continua a aumentar no mundo todo. De acordo com a Organização Mundial de Saúde, foram estimados 9,4 milhões de novos casos de TB em 2008, dos quais 1,4 milhões eram HIV - positivos, e com 1,8 milhões de mortes - o equivalente a 4.500 mortes por dia. Fatores como migração, privação sócio-econômica, co-infecção TB-HIV e o aparecimento de cepas resistentes contribuíram para o aumento do número de casos de TB no mundo, principalmente nos países onde a TB já foi considerada erradicada, e criaram a necessidade do desenvolvimento de novas terapêuticas. Alvos moleculares específicos, que são essenciais para o patógeno, e ausentes no hospedeiro, como as enzimas da via do ácido chiquímico são alvos atraentes para o desenvolvimento de novas drogas antituberculose. Essa via leva à síntese de compostos aromáticos, como aminoácidos aromáticos, e é encontrada em plantas, fungos, bactérias e parasitas do phylum Apicomplexa, mas está ausente em humanos. No ano de 2000, foi comprovada a essencialidade dessa via para a viabilidade do bacilo, tornando todas essas enzimas alvos validados para estudo. A segunda enzima da via, deidroquinato sintase (DHQS), catalisa a conversão de 3-deoxi-D-arabino heptulosonato-7-fosfato em 3-deidroquinato, o primeiro composto cíclico. Neste trabalho, são descritos o requerimento de metais divalentes na reação e a determinação do mecanismo cinético da DHQS. Os parâmetros cinéticos verdadeiros foram determinados e, juntamente com os experimentos de ligação, o mecanismo rápido-equilíbrio aleatório foi proposto. O tratamento com EDTA aboliu completamente a atividade de DHQS, sendo que a adição de Co+2 e Zn+2 levam a recuperação total e parcial da atividade enzimática, respectivamente. O excesso de Zn+2 inibe a atividade DHQS, e os dados de ITC indicaram a presença de dois sítios seqüenciais de ligação, o que é consistente com a existência de um sítio secundário inibitório. O protocolo de cristalização foi estabelecido e experimentos em andamento proporcionarão a elucidação da estrutura tridimensional da DHQS, que irá beneficiar tanto o desenho de novos inibidores como uma análise detalhada dos rearranjos do domínio da proteína. Em conjunto, estes resultados representam um passo essencial para o desenho racional de inibidores específicos que podem fornecer uma alternativa promissora para um novo, eficaz, e mais curto de tratamento para TB. / Although the estimated per capita tuberculosis (TB) incidence was stable in 2005, the number of new cases arising each year is still increasing globally. According with World Health Organization, there were estimated 9.4 million new TB cases in 2008, from which 1.4 million were HIV-positive, with 1.8 million deaths total – equal to 4500 deaths a day. Migration, socio-economic deprivation, HIV co-infection and the emergence of extensively-resistance strains, have all contributed to the increasing number of TB cases worldwide, mainly in countries where it was once considered eradicated, and have created an urgent need for the development of new therapeutics against TB. Specific molecular targets, that are essential to the pathogen, and absent in the host, like the enzymes of the shikimate pathway, are attractive targets to development of new antitubercular drugs. This pathway leads to the biosynthesis of aromatic compounds, including aromatic amino acids and it is found in plant, fungi, bacteria and Apicomplexa parasites, but is absent in humans. In 2000, this pathway was proved to be essential to the viability of the pathogen, which validates all its enzymes as potential targets. The second enzyme of this pathway, dehydroquinate synthase (DHQS), catalyzes the conversion of 3-deoxy-D-arabinoheptulosonate 7-phosphate in 3-dehydroquinate, the first cyclic compound. In this work, we described the metal requirement and kinetic mechanism determination of the dehydroquinate synthase. The determination of the true kinetic parameters was performed, and, in addition to ligand binding experiments, the rapid-equilibrium random mechanism was determined. The treatment with EDTA abolished completely the activity of DHQS, and the addition of Co+2 and Zn+2 leads to full and partial recovery of enzyme activity, respectively. Excess of Zn+2 inhibits the DHQS activity, and the ITC data revealed two sequential binding sites, which is consistent with the existence of a secondary inhibitory site. The crystallization protocol was established and ongoing experiments will provide the three-dimensional structure of mtDHQS, which will benefit both the design of novel inhibitors as well as detailed analysis of domain rearrangements of protein. Taken together, these results represent an essential step for the rational design of specific inhibitors that can provide a promising alternative to a new, effective, and shorter treatment for TB.

Apesar da incidência per capita da tuberculose (TB) ter se mantido estável em 2005, o número de novos casos que surgem a cada ano continua a aumentar no mundo todo. De acordo com a Organização Mundial de Saúde, foram estimados 9,4 milhões de novos casos de TB em 2008, dos quais 1,4 milhões eram HIV - positivos, e com 1,8 milhões de mortes - o equivalente a 4.500 mortes por dia. Fatores como migração, privação sócio-econômica, co-infecção TB-HIV e o aparecimento de cepas resistentes contribuíram para o aumento do número de casos de TB no mundo, principalmente nos países onde a TB já foi considerada erradicada, e criaram a necessidade do desenvolvimento de novas terapêuticas. Alvos moleculares específicos, que são essenciais para o patógeno, e ausentes no hospedeiro, como as enzimas da via do ácido chiquímico são alvos atraentes para o desenvolvimento de novas drogas antituberculose. Essa via leva à síntese de compostos aromáticos, como aminoácidos aromáticos, e é encontrada em plantas, fungos, bactérias e parasitas do phylum Apicomplexa, mas está ausente em humanos. No ano de 2000, foi comprovada a essencialidade dessa via para a viabilidade do bacilo, tornando todas essas enzimas alvos validados para estudo. A segunda enzima da via, deidroquinato sintase (DHQS), catalisa a conversão de 3-deoxi-D-arabino heptulosonato-7-fosfato em 3-deidroquinato, o primeiro composto cíclico. Neste trabalho, são descritos o requerimento de metais divalentes na reação e a determinação do mecanismo cinético da DHQS. Os parâmetros cinéticos verdadeiros foram determinados e, juntamente com os experimentos de ligação, o mecanismo rápido-equilíbrio aleatório foi proposto. O tratamento com EDTA aboliu completamente a atividade de DHQS, sendo que a adição de Co+2 e Zn+2 levam a recuperação total e parcial da atividade enzimática, respectivamente. O excesso de Zn+2 inibe a atividade DHQS, e os dados de ITC indicaram a presença de dois sítios seqüenciais de ligação, o que é consistente com a existência de um sítio secundário inibitório. O protocolo de cristalização foi estabelecido e experimentos em andamento proporcionarão a elucidação da estrutura tridimensional da DHQS, que irá beneficiar tanto o desenho de novos inibidores como uma análise detalhada dos rearranjos do domínio da proteína. Em conjunto, estes resultados representam um passo essencial para o desenho racional de inibidores específicos que podem fornecer uma alternativa promissora para um novo, eficaz, e mais curto de tratamento para TB. / Although the estimated per capita tuberculosis (TB) incidence was stable in 2005, the number of new cases arising each year is still increasing globally. According with World Health Organization, there were estimated 9.4 million new TB cases in 2008, from which 1.4 million were HIV-positive, with 1.8 million deaths total – equal to 4500 deaths a day. Migration, socio-economic deprivation, HIV co-infection and the emergence of extensively-resistance strains, have all contributed to the increasing number of TB cases worldwide, mainly in countries where it was once considered eradicated, and have created an urgent need for the development of new therapeutics against TB. Specific molecular targets, that are essential to the pathogen, and absent in the host, like the enzymes of the shikimate pathway, are attractive targets to development of new antitubercular drugs. This pathway leads to the biosynthesis of aromatic compounds, including aromatic amino acids and it is found in plant, fungi, bacteria and Apicomplexa parasites, but is absent in humans. In 2000, this pathway was proved to be essential to the viability of the pathogen, which validates all its enzymes as potential targets. The second enzyme of this pathway, dehydroquinate synthase (DHQS), catalyzes the conversion of 3-deoxy-D-arabinoheptulosonate 7-phosphate in 3-dehydroquinate, the first cyclic compound. In this work, we described the metal requirement and kinetic mechanism determination of the dehydroquinate synthase. The determination of the true kinetic parameters was performed, and, in addition to ligand binding experiments, the rapid-equilibrium random mechanism was determined. The treatment with EDTA abolished completely the activity of DHQS, and the addition of Co+2 and Zn+2 leads to full and partial recovery of enzyme activity, respectively. Excess of Zn+2 inhibits the DHQS activity, and the ITC data revealed two sequential binding sites, which is consistent with the existence of a secondary inhibitory site. The crystallization protocol was established and ongoing experiments will provide the three-dimensional structure of mtDHQS, which will benefit both the design of novel inhibitors as well as detailed analysis of domain rearrangements of protein. Taken together, these results represent an essential step for the rational design of specific inhibitors that can provide a promising alternative to a new, effective, and shorter treatment for TB.

Apesar da incidência per capita da tuberculose (TB) ter se mantido estável em 2005, o número de novos casos que surgem a cada ano continua a aumentar no mundo todo. De acordo com a Organização Mundial de Saúde, foram estimados 9,4 milhões de novos casos de TB em 2008, dos quais 1,4 milhões eram HIV - positivos, e com 1,8 milhões de mortes - o equivalente a 4.500 mortes por dia. Fatores como migração, privação sócio-econômica, co-infecção TB-HIV e o aparecimento de cepas resistentes contribuíram para o aumento do número de casos de TB no mundo, principalmente nos países onde a TB já foi considerada erradicada, e criaram a necessidade do desenvolvimento de novas terapêuticas. Alvos moleculares específicos, que são essenciais para o patógeno, e ausentes no hospedeiro, como as enzimas da via do ácido chiquímico são alvos atraentes para o desenvolvimento de novas drogas antituberculose. Essa via leva à síntese de compostos aromáticos, como aminoácidos aromáticos, e é encontrada em plantas, fungos, bactérias e parasitas do phylum Apicomplexa, mas está ausente em humanos. No ano de 2000, foi comprovada a essencialidade dessa via para a viabilidade do bacilo, tornando todas essas enzimas alvos validados para estudo. A segunda enzima da via, deidroquinato sintase (DHQS), catalisa a conversão de 3-deoxi-D-arabino heptulosonato-7-fosfato em 3-deidroquinato, o primeiro composto cíclico. Neste trabalho, são descritos o requerimento de metais divalentes na reação e a determinação do mecanismo cinético da DHQS. Os parâmetros cinéticos verdadeiros foram determinados e, juntamente com os experimentos de ligação, o mecanismo rápido-equilíbrio aleatório foi proposto. O tratamento com EDTA aboliu completamente a atividade de DHQS, sendo que a adição de Co+2 e Zn+2 levam a recuperação total e parcial da atividade enzimática, respectivamente. O excesso de Zn+2 inibe a atividade DHQS, e os dados de ITC indicaram a presença de dois sítios seqüenciais de ligação, o que é consistente com a existência de um sítio secundário inibitório. O protocolo de cristalização foi estabelecido e experimentos em andamento proporcionarão a elucidação da estrutura tridimensional da DHQS, que irá beneficiar tanto o desenho de novos inibidores como uma análise detalhada dos rearranjos do domínio da proteína. Em conjunto, estes resultados representam um passo essencial para o desenho racional de inibidores específicos que podem fornecer uma alternativa promissora para um novo, eficaz, e mais curto de tratamento para TB. / Although the estimated per capita tuberculosis (TB) incidence was stable in 2005, the number of new cases arising each year is still increasing globally. According with World Health Organization, there were estimated 9.4 million new TB cases in 2008, from which 1.4 million were HIV-positive, with 1.8 million deaths total – equal to 4500 deaths a day. Migration, socio-economic deprivation, HIV co-infection and the emergence of extensively-resistance strains, have all contributed to the increasing number of TB cases worldwide, mainly in countries where it was once considered eradicated, and have created an urgent need for the development of new therapeutics against TB. Specific molecular targets, that are essential to the pathogen, and absent in the host, like the enzymes of the shikimate pathway, are attractive targets to development of new antitubercular drugs. This pathway leads to the biosynthesis of aromatic compounds, including aromatic amino acids and it is found in plant, fungi, bacteria and Apicomplexa parasites, but is absent in humans. In 2000, this pathway was proved to be essential to the viability of the pathogen, which validates all its enzymes as potential targets. The second enzyme of this pathway, dehydroquinate synthase (DHQS), catalyzes the conversion of 3-deoxy-D-arabinoheptulosonate 7-phosphate in 3-dehydroquinate, the first cyclic compound. In this work, we described the metal requirement and kinetic mechanism determination of the dehydroquinate synthase. The determination of the true kinetic parameters was performed, and, in addition to ligand binding experiments, the rapid-equilibrium random mechanism was determined. The treatment with EDTA abolished completely the activity of DHQS, and the addition of Co+2 and Zn+2 leads to full and partial recovery of enzyme activity, respectively. Excess of Zn+2 inhibits the DHQS activity, and the ITC data revealed two sequential binding sites, which is consistent with the existence of a secondary inhibitory site. The crystallization protocol was established and ongoing experiments will provide the three-dimensional structure of mtDHQS, which will benefit both the design of novel inhibitors as well as detailed analysis of domain rearrangements of protein. Taken together, these results represent an essential step for the rational design of specific inhibitors that can provide a promising alternative to a new, effective, and shorter treatment for TB.

3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DHS) catalyzes the first step of the shikimate pathway - a pathway involved in Tyrosine (Tyr), Tryptophan (Trp) and Phenylalanine (Phe) biosynthesis - by condensation of phosphoenolpyruvate and erythrose-4-phosphate to DAHP. Our lab previously demonstrated that Arabidopsis thaliana shikimate pathway flux is regulated by Tyr and Trp. This project suggests that A. thaliana DHS1 overexpressor lines have increased Trp accumulation with Tyr treatment, and that an A. thaliana DHS2 overexpressor line treated with Tyr has unchanged Trp accumulation, indicating that AtDHS2 is Tyr-sensitive. Confocal microscopy of all 3 AtDHS isoforms fused to yellow fluorescent protein demonstrates chloroplast localization. Bimolecular fluorescence complementation indicates that protein-protein interactions occur in the cytoplasm, and not in the chloroplast, for AtDHS1 and AtDHS2 with the metabolic regulator At14-3-3ω. These findings suggest that protein-protein interactions could regulate accumulation of AtDHS2 in the chloroplast, and are perhaps modulated by Tyr.

3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DHS) catalyzes the first step of the shikimate pathway - a pathway involved in Tyrosine (Tyr), Tryptophan (Trp) and Phenylalanine (Phe) biosynthesis - by condensation of phosphoenolpyruvate and erythrose-4-phosphate to DAHP. Our lab previously demonstrated that Arabidopsis thaliana shikimate pathway flux is regulated by Tyr and Trp. This project suggests that A. thaliana DHS1 overexpressor lines have increased Trp accumulation with Tyr treatment, and that an A. thaliana DHS2 overexpressor line treated with Tyr has unchanged Trp accumulation, indicating that AtDHS2 is Tyr-sensitive. Confocal microscopy of all 3 AtDHS isoforms fused to yellow fluorescent protein demonstrates chloroplast localization. Bimolecular fluorescence complementation indicates that protein-protein interactions occur in the cytoplasm, and not in the chloroplast, for AtDHS1 and AtDHS2 with the metabolic regulator At14-3-3ω. These findings suggest that protein-protein interactions could regulate accumulation of AtDHS2 in the chloroplast, and are perhaps modulated by Tyr.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2017-09-01T13:05:01Z No. of bitstreams: 2 Dissertação - Wanderson Lucas da Costa - 2017.pdf: 3043089 bytes, checksum: 32c1b9c81dae778ab37d939fdba41eb5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-09-15T13:46:23Z (GMT) No. of bitstreams: 2 Dissertação - Wanderson Lucas da Costa - 2017.pdf: 3043089 bytes, checksum: 32c1b9c81dae778ab37d939fdba41eb5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-09-15T13:46:23Z (GMT). No. of bitstreams: 2 Dissertação - Wanderson Lucas da Costa - 2017.pdf: 3043089 bytes, checksum: 32c1b9c81dae778ab37d939fdba41eb5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-08-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Paracoccidioides spp. are thermodymorphic fungi that when inhaled by humans, these conidia find a favorable environment, changing to the yeast phase and becoming pathogenic causing paracoccidioidomycosis (PCM), one of the most prevalent systemic mycoses in Brazil. Some antifungals are used in the treatment of PCM. Treatment depends on the patient's progression and tolerability of each drug, but their treatment may be for long periods and cause various side effects in the patient. The chiquimate pathway is coordinated by 7 enzymes that perform consecutive steps to convert erythrose-4-phosphate and phosphoenol pyruvate (PEP) into chorismate. In microorganisms, this pathway is involved in the production of the amino acids phenylalanine, tyrosine and tryptophan; These amino acids are essential to the maintenance of these organisms. In this work, pGEX4T3 vector cloning and heterologous expression of Pb18 EPSP synthase belonging to the chiquimate pathway were performed. This protein was expressed in E. coli (DE3) strain and purified. Antibodies were produced for expression analysis of the protein in Western blot. The modeling of EPSP synthase was performed aiming to identify the amino acids involved in the active site. The pull down-GST assay with soluble Pb18 proteins allowed the identification of 40 proteins that interact with EPSP synthase. These proteins belong to different functional categories, which are involved with the availability of phosphoenol pyruvate, the substrate necessary for the functioning of the chiquimate pathway. / Paracoccidioides spp. são fungos termodimórficos que ao serem inalados pelo ser humano, esses conídios encontram um ambiente propício, mudando para a fase de levedura e tornando-se patogênico causando a paracoccidioidomicose (PCM), umas das micoses sistêmicas de maior prevalência no Brasil. Alguns antifúngicos são empregados no tratamento da PCM. O tratamento depende do avanço da doença e da capacidade de tolerância do paciente a cada medicamento, mas o seu tratamento pode ser por longos períodos e causando diversos efeitos colaterais no paciente. A via do chiquimato é coordenada pela ação de 7 enzimas que realizam passos consecutivos para transformar a eritrose-4-fosfato e fosfoenol piruvato (PEP) em corismato. Em micro-organismos, esta via está envolvida com a produção dos aminoácidos fenilalanina, tirosina e triptofano; estes aminoácidos são essenciais à manutenção desses organismos. Neste trabalho foi realizado a clonagem em vetor pGEX4T3 e expressão heteróloga da EPSP–sintase de Pb18 pertencente à via do chiquimato. Essa proteína foi expressa em linhagem E. coli (DE3) e purificada. Os anticorpos foram produzidos para análise da expressão da proteína em Western blot. A modelagem da EPSP-sintase foi realizada visando identificar os aminoácidos envolvidos no sítio ativo. O ensaio de pull down-GST com proteínas solúveis de Pb18 possibilitou a identificação de 40 proteínas que interagem com EPSP-sintase. Essas proteínas pertencem a diferentes categorias funcionais, as quais estão envolvidas com a disponibilidade de fosfoenol piruvato, substrato necessário para o funcionamento da via do chiquimato.

3-Deoxy-D-arabino heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first step of the shikimate pathway, responsible for the biosynthesis of aromatic amino acids. This pathway is found in microorganisms, plants and apicomplexan parasites and its absence in mammals makes it a viable target for antimicrobial drug design. DAH7PS enzymes differ in the regulatory machinery that decorates the catalytic (β/α)8 barrel. Some DAH7PS enzymes are fused to chorismate mutase (CM), another enzyme in the shikimate pathway. This fusion protein is allosterically regulated by chorismate (CA) or prephenate (PA), the precursor of tyrosine and phenylalanine. It has been suggested that DAH7PS enzymes evolved these extensions to the core barrel for the sole purpose of regulation. Geobacillus sp DAH7PS (GspDAH7PSWT) is a thermophilic type Iβ DAH7PS enzyme with an N-terminal CM domain fused through a linker region. This thesis describes the functional characterisation work carried out on GspDAH7PSWT, in attempt to help determine how DAH7PS enzymes evolved such diverse methods of regulation. Chapter 2 describes the functional characterisation work carried out on the catalytic and regulatory domains of GspDAH7PSWT. The enzyme demonstrated both DAH7PS and CM activities with the DAH7PS domain determined to be metal dependent and most activated by Cd2+. PA completely inhibited the catalytic activity of GspDAH7PSWT, and AUC demonstrated an equilibrium exists between the dimeric and tetrameric quaternary states of the enzyme in solution. Chapter 3 describes the domain truncation of GspDAH7PSWT carried out at the linker region in order to obtain two separate protein domains, the catalytic domain lacking the N-terminal domain (GspDAH7PSDAH7PS) and the regulatory domain without the catalytic domain (GspDAH7PSCM). Both variants were fully characterised, and information obtained from each domain was compared to the respective catalytic and regulatory domains of the wild-type enzyme, which was also characterised. Like GspDAH7PSWT, GspDAH7PSDAH7PS showed greatest activation in the presence of Cd2+, with other metals having varying effects on activation rates and stability of the enzyme. Both truncated variants followed Michaelis-Menten kinetics where GspDAH7PSDAH7PS was found to be more active than GspDAH7PSWT and unaffected by PA, whereas GspDAH7PSCM was a less efficient catalyst than the CM domain of GspDAH7PSWT. AUC demonstrated that in solution an equilibrium occurs between the monomeric and tetrameric oligomeric states of GspDAH7PSDAH7PS. Chapter 4 summarises the findings of the thesis along with future directions of this research, combining the results obtained and expanding upon them. It is concluded that the catalytic regulatory CM domain supports both protein structure and allosteric regulation of GspDAH7PSWT

3-Deoxy-D-arabino heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first step of the shikimate pathway, responsible for the biosynthesis of aromatic amino acids. This pathway is found in microorganisms, plants and apicomplexan parasites and its absence in mammals makes it a viable target for antimicrobial drug design. DAH7PS enzymes differ in the regulatory machinery that decorates the catalytic (β/α)8 barrel. Some DAH7PS enzymes are fused to chorismate mutase (CM), another enzyme in the shikimate pathway. This fusion protein is allosterically regulated by chorismate (CA) or prephenate (PA), the precursor of tyrosine and phenylalanine. It has been suggested that DAH7PS enzymes evolved these extensions to the core barrel for the sole purpose of regulation. Geobacillus sp DAH7PS (GspDAH7PSWT) is a thermophilic type Iβ DAH7PS enzyme with an N-terminal CM domain fused through a linker region. This thesis describes the functional characterisation work carried out on GspDAH7PSWT, in attempt to help determine how DAH7PS enzymes evolved such diverse methods of regulation. Chapter 2 describes the functional characterisation work carried out on the catalytic and regulatory domains of GspDAH7PSWT. The enzyme demonstrated both DAH7PS and CM activities with the DAH7PS domain determined to be metal dependent and most activated by Cd2+. PA completely inhibited the catalytic activity of GspDAH7PSWT, and AUC demonstrated an equilibrium exists between the dimeric and tetrameric quaternary states of the enzyme in solution. Chapter 3 describes the domain truncation of GspDAH7PSWT carried out at the linker region in order to obtain two separate protein domains, the catalytic domain lacking the N-terminal domain (GspDAH7PSDAH7PS) and the regulatory domain without the catalytic domain (GspDAH7PSCM). Both variants were fully characterised, and information obtained from each domain was compared to the respective catalytic and regulatory domains of the wild-type enzyme, which was also characterised. Like GspDAH7PSWT, GspDAH7PSDAH7PS showed greatest activation in the presence of Cd2+, with other metals having varying effects on activation rates and stability of the enzyme. Both truncated variants followed Michaelis-Menten kinetics where GspDAH7PSDAH7PS was found to be more active than GspDAH7PSWT and unaffected by PA, whereas GspDAH7PSCM was a less efficient catalyst than the CM domain of GspDAH7PSWT. AUC demonstrated that in solution an equilibrium occurs between the monomeric and tetrameric oligomeric states of GspDAH7PSDAH7PS. Chapter 4 summarises the findings of the thesis along with future directions of this research, combining the results obtained and expanding upon them. It is concluded that the catalytic regulatory CM domain supports both protein structure and allosteric regulation of GspDAH7PSWT