The shikimate pathway is responsible for the biosynthesis of essential aromatic metabolites and, as such, its enzymes are targets for the design of potential antimicrobial and herbicidal agents. The enzyme 3-dehydroquinate dehydratase (dehydroquinase, DHQase) catalyses the conversion of 3-dehydroquinate to 3-dehydroshikimate, the third step of the shikimate pathway. There are two types of DHQase, unrelated structurally and mechanistically. Type I DHQase catalyses the rection by via a covalently attached imine intermediate. Type II DHQase catalyses the reaction by way of an enolate intermediate. This thesis describes the synthesis of a series of potential inhibitors of type II DHQase. Inhibitors with C and N at C-3 and with both sp2 and sp3 character at this position were prepared. A potential type I DHQase inhibitor was also prepared. The biological evaluation of these inhibitors against type II DHQases from Mycobacterium tuberculosis and Streptomyces coelicolor and type I DHQase from Salmonella typhi is described. Inhibitors were evaluated by spectrophotometric assay. However, this proved inappropriate for some inhibitors with the S. coelicolor enzyme. The development of an alternative 1H NMR assay and its application to the evaluation of S. coelicolor DHQase inhibitors is therefore also described. Some insights into the structure activity relationships of type II DHQases, obtained from the results of these studies, are discussed.

Allosteric regulation of important enzymes is a mechanism frequently employed by organisms to exert control over their metabolism. The shikimate pathway is ultimately responsible for the biosynthesis of the aromatic amino acids in plants, microorganisms and apicomplexans. Two enzymes of the pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) and chorismate mutase (CM) are located at critical positions along the aromatic amino acid biosynthetic pathway and are often tightly feedback regulated in order to control the flux of metabolites through the pathway. This research presents studies on the allosteric function of these two enzymes. These studies emphasise the complexity of the intersecting network of allosteric response, which alters the catalytic activity of each enzyme in response to metabolic demand for the aromatic amino acids.

The shikimate pathway is responsible for the biosynthesis of the aromatic amino acids and other aromatic metabolites in plants, micro-organisms and apicomplexan parasites. The shikimate pathway is essential in bacteria and plants, but absent from mammals, which has led to interest in the enzymes of the pathway as targets for the design of antimicrobial and herbicidal agents. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first commit¬ted step of the shikimate pathway, the condensation of phosphoenolpyruvate and erythrose 4-phosphate to yield 3-deoxy-D-arabino-heptulosonate 7-phosphate. The subject of this thesis is the investigation of inhibition and allosteric regulation of the DAH7PS enzyme from Myco¬bacterium tuberculosis (MtuDAH7PS), the pathogen that causes tuberculosis. Tuberculosis remains a major health threat to the global community, and the emergence of multi-drug resistant strains highlights the need for new tuberculosis treatments. Inhibitors of MtuDAH7PS have the potential to be developed into new anti-tuberculosis drugs. Chapter 2 describes the design, synthesis and evaluation of active site inhibitors based on intermediate mimic scaffolds. The intermediate mimics synthesised represent the first reported example of inhibitors targeting the active site of MtuDAH7PS. The most active compounds tested displayed inhibition constants in the sub-micromolar range, making them the most potent inhibitors of any DAH7PS enzyme reported to date. MtuDAH7PS displays a complex and subtle mechanism of synergistic regulation: The enzyme is inhibited by binary combinations of the aromatic amino acids tryptophan (Trp), phenylalanine (Phe) and tyrosine (Tyr). Three allosteric binding sites were identified using X-ray crystallo¬graphic analysis of MtuDAH7PS in complex with Trp and Phe. While these crystal structures led to the identification of an allosteric binding site which preferentially binds Trp, the role and selectivity of the other two sites with respect to Phe and Tyr remained unclear. The results described in Chapter 3 provide structural and biochemical evidence for the hypothesis that each of the three allosteric binding sites has a preference for binding one of the aromatic amino acids Trp, Phe and Tyr, respectively. The results furthermore show that the ternary combination of Trp, Phe and Tyr synergistically regulates MtuDAH7PS, leading to almost complete loss of enzymatic activity in the presence of all three allosteric ligands. In Chapter 4, the interaction of MtuDAH7PS with the naturally less common D-enantiomers of the aromatic amino acids is described. It was found that the D-enantiomers of the aromatic amino acids have no effect on enzymatic activity of MtuDAH7PS, suggesting an efficient mechanism by which the enzyme can discriminate between allosteric ligands of opposite configuration. Studies of the binding affinity of the D-amino acids to MtuDAH7PS as well as structural characterisation of MtuDAH7PS-D-amino acid complexes by X-ray crystallographic analysis suggest that D-Trp and D-Phe can still bind to their respective sites. The lack of inhibition is attributed to subtle differences in the binding mode of the D-enantiomers of the ligands compared to the L-enantiomers. Chapter 5 details the discovery of alternative ligands and inhibitors targeting the allosteric sites of MtuDAH7PS using virtual screening. Libraries of potential alternative ligands were docked into the allosteric sites of MtuDAH7PS and the predicted docking poses were used to guide the selection of compounds for physical screening. Using this approach, a number of ligands and inhibitors of MtuDAH7PS were discovered and their interaction with the enzyme structurally characterised. Comparison of the crystallographically observed binding modes of new ligands with the docking poses predicted by computational docking highlighted potential improvements to the virtual screening method. The analysis of the correlation between ligand binding modes and inhibition of enzymatic activity provided further insight into which interactions between the allosteric ligand and the binding site are crucial in order to achieve inhibition. The crystal structures of MtuDAH7PS in complex with the new alternative ligands can serve as a starting point for the design of ligands with increased affinity and potency.

The shikimate pathway is responsible for the biosynthesis of essential aromatic metabolites and, as such, its enzymes are targets for the design of potential antimicrobial and herbicidal agents. The enzyme 3-dehydroquinate dehydratase (dehydroquinase, DHQase) catalyses the conversion of 3-dehydroquinate to 3-dehydroshikimate, the third step of the shikimate pathway. There are two types of DHQase, unrelated structurally and mechanistically. Type I DHQase catalyses the rection by via a covalently attached imine intermediate. Type II DHQase catalyses the reaction by way of an enolate intermediate. This thesis describes the synthesis of a series of potential inhibitors of type II DHQase. Inhibitors with C and N at C-3 and with both sp2 and sp3 character at this position were prepared. A potential type I DHQase inhibitor was also prepared. The biological evaluation of these inhibitors against type II DHQases from Mycobacterium tuberculosis and Streptomyces coelicolor and type I DHQase from Salmonella typhi is described. Inhibitors were evaluated by spectrophotometric assay. However, this proved inappropriate for some inhibitors with the S. coelicolor enzyme. The development of an alternative 1H NMR assay and its application to the evaluation of S. coelicolor DHQase inhibitors is therefore also described. Some insights into the structure activity relationships of type II DHQases, obtained from the results of these studies, are discussed.

The shikimate pathway is a biosynthetic pathway that is responsible for producing a variety of organic compounds that are necessary for life in plants and microorganisms. The pathway consists of seven enzyme catalysed reactions beginning with the condensation reaction between D-erythrose 4-phosphate (E4P) and phosphoenolpyruvate (PEP) to give the seven-carbon sugar DAH7P. This thesis describes the design, synthesis and evaluation of a range of alternative non-natural four-carbon analogues of E4P (2- and 3-deoxyE4P, 3-methylE4P, phosphonate analogues of E4P) to probe the substrate specificity of different types of DAH7P synthases [such as Mycobacterium tuberculosis (a type II DAH7PS), Escherichia coli (a type Ialpha DAH7PS) and Pyrococcus furiosus (a type Ibeta DAH7PS)].

Content removed due to copyright restrictions: Webby, C.J., Patchett, M.L. & Parker, E.J. (2005) Characterization of a recombinant type II 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Helicobacter pylori. Biochemical Journal 390, 223-230 Webby C.J., Lott J.S., Baker H.M., Baker E.N., & Parker E.J. (2005) Crystallization and preliminary X-ray crystallographic analysis of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Mycobacterium tuberculosis. Acta Crystallographica Section F - Sturctural Biology and Crystallization Communications 61(4) 403-406. Webby C.J., Baker H.M., Lott J.S., Baker E.N. & Parker E.J. (2005) The structure of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Mycobacterium tuberculosis reveals a common catalytic scaffold and ancestry for type I and type II enzymes. Journal of Molecular Biology 354(4), 927-939 / The shikimate pathway, responsible for the biosynthesis of aromatic compounds, is found in microorganisms and plants but absent in higher organisms. This makes the enzymes of this pathway attractive as targets for the development of antibiotics and herbicides. Recent gene disruption studies have shown that the operation of the shikimate pathway is essential for the viability of M. tuberculosis, validating the choice of enzymes from this pathway as targets for the development of novel anti-TB drugs. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first committed step of the shikimate pathway. Two distinct classes of DAH7PS have been defined based on sequence similarity. The type I DAH7PSs are well characterized, however prior to this project there was limited mechanistic and no structural information about type II enzymes. Sequence identity between type I and type II enzymes is less than 10% raising the possibility that they represent distinct protein families, unrelated by evolution. We have functionally characterized the type II enzyme from Helicobacter pylori, and have shown that type I and type II enzymes catalyze a metal-dependent ordered sequential reaction following the same stereochemical course. We have solved the structure of the type II DAH7PS from M. tuberculosis using single-wavelength anomalous diffraction (SAD) methods and the structure reveals a tightly associated dimer of (β/α)8 TIM barrels. The monomer fold, the arrangement of key residues in the active site, and the binding modes of PEP and Mn2+, all match those of the type I enzymes. This similarity of protein fold and catalytic architecture makes it unequivocal that type I and type II enzymes are related by divergent evolution from a common ancestor. Interestingly, there are significant differences in the additional structural elements that extend from the core (β/α)8 barrel and in the quaternary structure. Further structural and functional analysis of M. tuberculosis DAH7PS revealed that the two major additions decorating the barrel are involved in the binding of the aromatic amino acids. Two distinct inhibitory binding sites for Trp and Phe have been identified providing an explanation for the synergistic inhibition displayed with Trp and Phe. The role of several active site residues of Mt-DAH7PS in enzyme catalysis has also been investigated.

<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.

Les plantes produisent une grande variété de produits naturels pour faire face aux conditions environnementales. Les enzymes de la famille CYP98 des cytochromes P450 sont des enzymes clés dans la production des composés dérivés de la voie des phénylpropanoïdes. Ces enzymes sont impliquées dans l'hydroxylation des esters phénoliques pour la biosynthèse des monolignols chez les angiospermes, mais elles sont également impliquées dans la production de divers autres composés phénoliques solubles. Nous avons caractérisé des CYP98 représentatifs des mousses, Lycopodes, fougères, Gymnospermes, Angiospermes basales, Monocotylédones et Eudicotylédones et démontré que leur préférence de substrat a changé au cours de l'évolution. Un mutant knock-out de CYP98 de mousse a révélé un phénotype sévère et que le p-coumaroyl-thréonate est substrat de l’enzyme in vivo. Une duplication des CYP98s ne peut être observée que dans le génome des Angiospermes, qui présentent généralement une isoforme potentiellement impliquée dans la biosynthèse de la lignine et autres isoformes, résultant de duplications indépendantes, dont le spectre de substrats est plus large in vitro. / Plants produce a rich variety of natural products to face environmental constraints. Enzymes of the cytochrome P450 CYP98 family are key actors in the production of phenolic bioactive compounds. They hydroxylate phenolic esters for lignin biosynthesis in angiosperms, but also produce various other bioactive phenolics. We characterized CYP98s from a moss, a lycopod, a fern, a conifer, a basal angiosperm, a monocot and from two eudicots. We found that substrate preference of the enzymes has changed during evolution of land plants with typical lignin-related activities only appearing in angiosperms, suggesting that ferns, similar to lycopods, produce lignin through an alternative route. A moss CYP98 knock-out mutant revealed coumaroyl-threonate as CYP98 substrate in vivo and showed a severe phenotype. Multiple CYP98s per species exist only in the angiosperms, where we generally found one isoform presumably involved in the biosynthesis of monolignols, and additional isoforms, resulting from independent duplications, with a broad range of functions in vitro.

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.