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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 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.

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 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 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.