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In fungi the central five steps of the shikimate pathway are fused into a single protein called the AROM enzyme. This has been shown to be related to the QUTA and QUTR proteins found in fungi which are responsible for the activation and repression of the transcription of enzymes found in the quinic acid utilisation pathway. The shikimate and quinate pathways therefore appear to be linked by a complex web of evolutionary relationships. Here research attempting to understand more fully the active mechanism of type I dehydroquinase from <i>S. typhi</i> is discussed. This research utilises molecular biological, biochemical and crystallographic techniques. Crystallographic trials of the dehydroquinase like domain of QUTR from <i>A. Nidulans</i> are also discussed. After literature review in chapter 1 and explanation of experimental techniques used in chapter 2, chapter 3 describes the production of active site mutants of type I dehydroquinase from <i>S. typhi</i>. These mutants are shown to decrease enzyme activity. In chapter 4 crystallisation of type I dehydroquinanse and its subsequent diffraction is described and discussed. Chapter 5 describes and discusses five different forms of wild type I dehydroquinase from <i>S. typhi</i>. In chapter 6 mutant crystal structures are described and discussed. In chapter 7 kinetic studies of putative dehydroquinase inhibitors are described and discussed. The crystal structure of one of these inhibitors is also described. In chapter 8 crystallisation trials of the dehydroquinase like domain of the QutR protein are discussed. In chapter 9 all results are discussed including the unusual packing of type I dehydroquinase from <i>S. typhi</i> and the implications of results to explaining the enzymes reaction mechanism. A reaction mechanism for type I dehydroquinase is proposed. In appendix 1 the structural solution and refinement of cytochrome C4 from <i>Pseudomonas aeruginosa</i> is described and compared to a previously solved cytochrome C4 from <i>Pseudomonas stutzeri.</i>

Efficient acquisition of the genes (<I>hsdR, M</I> and <I>S</I>) that specify <I>Eco</I>KI and <I>Eco</I>AI, representatives of two families of type I restriction and modification (R-M) systems, was shown to require a product of an unknown gene <I>hsd</I>C. The <I>hsd</I>C mutant is shown to have a mutation in <I>clp</I>X. ClpP, the components of ClpXP protease, are necessary for the efficient transmission of the <I>hsd</I> genes by conjugation, transformation and P1 transduction. Inactivation of <I>clp</I>X leads to a bigger barrier than a similar mutation in <I>clpP</I> consistent with a chaperone activity of ClpX in the absence of ClpP. The establishment of the modification activity of <I>Eco</I>KI is not dependent on <I>clp</I>X and takes about 12 generations to reach its maximal activity in methylating incoming phage DNA. This lag probably reflects the time necessary to complete the methylation of bacterial chromosomes. Modification, once established, has been assumed to provide adequate protection against a resident restriction system. However, unmodified targets may be generated in the DNA of an <I>hsd<SUP>+</SUP> </I>bacterium as the result of replication errors or recombinant-dependent repair. The presence of unmodified target sequences for type I restriction-modification systems on bacterial chromosomes does not influence the survival of <I>hsd<SUP>+</SUP> </I>bacteria due to ClpXP- dependent regulation of the endonuclease activity. HsdR, the polypeptide of the R-M complex essential for restriction but not modification, is degraded in the presence of ClpXP and therefore the bacteria show a temporary drop in restriction activity, referred to as restriction alleviation. The delayed detection of restriction activity followed by the establishment of a new specificity can be considered as a case of restriction alleviation. The data obtained support a model in which the HsdR component of a type I restriction endonuclease becomes a substrate for proteolysis after the endonuclease has bound to unmodified target sequences on the chromosome, but before completion of the pathway that would result in DNA breakage. It remains unclear how the restriction-modification systems distinguish between unmethylated host and foreign DNA. The latter is degraded while the former is protected from cleavage by ClpXP-dependent proteolysis of HsdR.

Ubiquitin and ubiquitin-like proteins (Ubls) are conjugated to proteins to regulate activity, stability, localisation or function. Ubiquitin/Ubl conjugation pathways are highly conserved in eukaryotes, and usually involve activating enzymes (E1s) and conjugating enzymes (E2s) specific for each ubiquitin/Ubl. Many studies have suggested that ubiquitin/Ubl conjugation pathways are important for oxidative stress resistance. However, there is much to learn regarding the roles of these pathways in oxidative stress responses. Additionally, limited studies in mammalian cells and yeast have indicated that certain E1s and E2s are redox-regulated, although how this relates to stress resistance is largely unclear, and these regulatory mechanisms have never been shown to be conserved in eukaryotes. Here, the roles and regulation of ubiquitin/Ubl conjugation pathways in responses to oxidative stress are investigated in Schizosaccharomyces pombe. Firstly, while our previous research has shown that the budding yeast E2, Cdc34, is redox-regulated, it was unclear whether ubiquitination is redox-regulated in other organisms. Results presented here show that the fission yeast Cdc34 homologue, Ubc15, is redox-regulated, suggesting that redox regulation of specific ubiquitination events may be conserved. Furthermore, Ubc15 is important for resistance to oxidative stress in S. pombe. Secondly, the Ubl Urm1 is found to be important for resistance to a range of stress conditions in S. pombe, as in S. cerevisiae, thus demonstrating for the first time that urmylation has conserved roles in stress resistance in eukaryotes. Additionally, urmylation controls the activation of a conserved mitogen-activated protein kinase during exposure to H2O2. Finally, although autophagic Ubl conjugation is not important for oxidative stress responses in S. pombe, these investigations have identified an E2 with roles in oxidative stress responses and cell cycle control. Taken together, these findings advance the study of the roles of ubiquitin/Ubl conjugation pathways in responses to oxidative stress, and offer exciting prospects for future investigations.

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