The type I restriction enzyme <i>Eco</i>KI specifies DNA methyltransferase, ATPase, DNA translocase and endonuclease activities. One subunit (HsdR) of the oligomeric enzyme contributes to those activities essential for restriction. These activities are thought to involve ATP-dependent DNA translocation and DNA cleavage. Analyses of recently predicted amino acid sequences of known and putative type I restriction endonucleases indicates conservation of eight amino acid motifs in the HsdR subunit. Seven are characteristics of the DEAD-box family of proteins that comprise known, or putative, helicases whilst the eighth was identified as a putative endonuclease motif due to its similarity to the active sites of type II restriction endonucleases and other nucleases. Secondary structure predictions based on sequences alignments of HsdR sequences suggest the DEAD-box motifs reside in domains similar to the catalytic domains of DNA helicases of known structure. Mutations affecting each of the DEAD-box motifs, including a new candidate for motif IV, impair or abolish restriction activity <i>in vivo</i> and ATPase activity <i>in vitro</i> (Webb <i>et al</i>., 1996; Webb, 1998; Davies <i>et al.,</i> 1998). Alteration of conserved residues in the putative endonuclease motif resulted in complete loss of restriction <i>in vivo</i> and endonuclease activity <i>in vitro</i>. Enzyme purified from two of these mutants, those with the alterations D298E and E312H, bound DNA with an <i>Eco</i>KI target and hydrolysed ATP at rates equivalent to wild-type on covalently-closed circular DNA with unmodified targets without introducing any nicks or breaks into the DNA. To investigate the role of conserved motifs in HsdR on DNA translocation more directly an assay based on the <i>Eco</i>KI-dependent entry of T7 DNA was used (Garcia and Molineux, 1999). Mutations within the seven DEAD-box motifs abolished translation activity <i>in vivo</i> whereas conservation mutations in the putative endonuclease motif had no significant effect on DNA translocation <i>in vivo</i>.

No description available.

CPT I was localised in the outer mitochondrial membrane and then partially purified. The protein was digested with restriction endonucleases and the fragments N-terminally sequenced and used to generate oligonucleotides for cDNA library screening and polymerase chain reaction (PCR). The partially purified protein was also used to generate polyclonal antibodies, which were used to screen λgt11 cDNA libraries. A positive clone was isolated from the λgt11 cDNA library using affinity purified serum. When sequenced, the clone was identified as long chain fatty acid CoA synthase. CPT II was purified from rat liver mitochondrial inner membranes and n-terminally sequenced. The purified protein was used to generate polyclonal antibodies. Oligonucleotides were generated to the cDNA encoding CPT II reported by Woeltje <I>et al</I> (1990b) and used to PCR up a 300 bp fragment, which when cloned and sequenced was identified as the C-terminus of CPT II. This fragment was then used unsuccessfully to screen λgt10, λgt11 and λgt11-stretch cDNA libraries, in order to find a full length clone. Using the information generated by this thesis along with other recent publications a possible topology of CPT I in the rat liver mitochondrial outer membrane was elucidated.

The intra-molecular electron transfer steps in three multi-centred oxidoreductases were studied by redox potentiometry and protein film voltammetry. Cytochrome <i>cd₁ </i>is a disimmilatory nitrite reductase, the achieves the reduction of nitrite to nitric <i>oxide.</i> Reduction of the enzyme from <i>Thiosphaera pantotropha</i> is associated with a conformational change (Williams <i>et al.,</i> 1997) and hysteresis is observed in the potentiometric titration at room temperature (Kobayashi <i>et al.,</i> 1997). The potentials in the oxidative and reductive directions are separated by 150 mV at 20°C but ?DE decreases at high temperatures. The increased reversibility at higher temperatures is also shown by a rise in the n-value and the appropriate spectral response when the potential is perturbed in both the oxidative and reductive directions. At the highest temperatures, when equilibrium conditions are reached, the interconversion of the completely oxidised and completely reduced states is characterised by a potential of 140 mV and an n-value of 2. At 20 °C the electron transfer is conformationally gated such that a proposed square scheme is essentially a one-way system. As the temperature is raised, the rate of the conformational change increases and the interconversion approaches reversibility within the measurement time. Flavocytochrome P-450 BM3 is a fatty-acid monooxygenase from <i>Bacillus megaterium.</i> The complete redox characterisation of the enzyme has been achieved by potentiometric titrations on the intact holoenzyme and its component domains. The reduction potential of the haem is increased by more than 100 mV on fatty-acid substrate binding at the active site (E_m = -368 ± 6 mV, substrate-free, to E_m = -239 ± 6 mV, arachidonate-bound). The FMN to haem electron-transfer is under thermodynamic control, such that the futile cycling of electrons and hydrogen peroxide formation is prevented. The blue FAD semiquinone species is observed during the course of the reductive titrations of these enzymes (E₁ = -283 mV).

The functional <i>mariner</i> element <i>Mos1 </i>is 1286 bp long with 28 bp imperfect terminal repeats and contains a single open reading frame encoding a 345 amino acid transposase. In this thesis, the N-terminal domain of mariner transposase was found to bind specificity to inverted repeats at both ends. The minimal DNA-binding domain was identified to be located between amino acid 1 and 120. This contains a helix-turn-helix (HTH) motif. Site-directed mutagenesis indicates that the HTH is required for sequence-specific recognition of the terminal inverted repeats by Mos1 transposase. The transposase has 5 to 6 times higher affinity for the right end sequence of Mos1 than for the left end. Yeast two-hybrid assays were performed to identify protein-protein interactions of Mos1 transposase. Deletion mutants suggest that residues required for the interaction of transposase monomers are distributed along the length of the protein. Twelve single point mutations which reduce subunit interactions have been isolated. The L124S mutation is located in neither the DNA-binding nor the catalytic domain. The purified Mos1(L124S) mutant reduces transposase activity in both excision and transposition assay, indicating that subunit interactions are involved in the transposition reaction. A change in target site selection other than TA dinucleotide observed with this mutant may reflect a change in the conformation of the transpososome. The promoter activity of <i>Mos1</i> was detected in transgenic flies by <i>P</i> element-mediated germline transformation. The promoter of <i>Mos1 </i>lies within nucleotides 1-171 which shows low activity. There is an enhancer-like element in the region of nucleotides 172-516 that stimulates reporter gene expression. No autoregulatory inhibition of Mos1 transposase on its own promoter was observed. This suggests that "overproduction inhibition" may not act at the level of transcription through autorepression of the <i>Mos1 </i>promoter.

The self sufficient class III monooxygenase, flavocytochrome P450 BM3, from <i>Bacillus megaterium</i>, is a heme containing redox enzyme that catalyses the hydroxylation of long-chain fatty acids. Flavocytochrome P450 BM3 contains several active-site residues that are highly conserved throughout the P450 superfamily. Of these, a phenylalanine (Phe393) has been shown to modulate the heme reduction potential (E_m) across a 200 mV range via interactions with the implicitly conserved heme-ligand cysteine. In addition, a distal threonine (Thr268) has been implicated in a variety of roles including proton donation, oxygen activation and substrate recognition. Substrate binding in P450 BM3 causes a shift in the spin state from low- to high-spin. This change in spin-state is accompanied by a positive shift in the reduction potential (ΔE_m[WT + arachidonate (120 μM)] = +138 mV). Substitution of Thr268 by alanine or asparagines causes a significant decrease in the ability of the enzyme to generate the high-spin complex via substrate binding and consequently leads to a decrease in the substrate-induced potential shift (ΔE_m[T268A + arachidonate (120 μM)] = +73 mV, ΔE_m {T268N + arachidonate (120 μM)] = +9 mV). Rate constants for the first electron transfer and for oxy-ferrous decay were measured by pre-steady state stopped-flow kinetics and found to be almost entirely dependant on the heme reduction potential. More positive reduction potentials lead to enhanced rate constants for heme reduction and more stable oxy-ferrous species. These results suggest an important role for this active-site threonine in substrate recognition and in maintaining an efficiently functioning enzyme.

Enantiomerically pure chiral secondary alcohols are important intermediates in the fine chemical, pharmaceutical and agrochemical industries. Many processes exist for generating enantiomerically pure compounds. One of these processes, deracemisation, functions by the combination of repeated cycles of enantioselective enzymatic oxidation and non-specific chemical reduction to convert a racemic mixture to a single enantiomer. The aim of this project was to provide an effective secondary alcohol oxidase for the oxidation step of deracemisation processes. To this end, poly(vinyl alcohol) oxidase from <i>Brevundimonas vesicularis</i> was chosen due to its broad substrate specificity. A purification method was developed and assays carried out establishing its preference for <i>R</i>-secondary alcohols. Due to the slow growth of <i>B. vesicularis,</i> it was decided to clone the gene encoding PVA oxidase. The goal was to develop a robust biocatalysed for industrial applications; therefore, rapid and reliable access to large quantities of the enzyme was necessary. This could be provided by an over-expressed recombinant PVA oxidase. Attempts at cloning were made with Southern hybridisation, inverse PCR, and analysis of an expression library. Much forward progress was made, but the gene remained elusive. Subsequently, the use of crude PVA oxidase preparations was investigated. These have the potential to quickly and practically oxidise a variety of chiral secondary alcohols.

DNA ligases join breaks in double-stranded DNA and are therefore crucial enzymes in all aspects of DNA metabolism. Eukaryotic DNA ligase I homologues belong to the family of ATP-dependent ligases and join Okazaki fragments generated on the lagging strand during chromosomal DNA replication. DNA ligase I enzymes consist of C-terminal catalytic domains conserved across all ATP-dependent ligases, a middle conserved domain of unknown function and N-terminal extensions, which share a conserved PCNA binding motif, but otherwise show very limited sequence similarity. In this work structure-function analyses on the DNA ligase I homologue Cdc17 of <i>Schizosaccharomyces pombe </i>were performed. The presence of the middle conserved non-catalytic domain in addition to the catalytic domains was found to be essential for rescue of a <i>cdc17</i> deletion strain. The N-terminal domain targets the enzyme to the nucleus and mitochondria and essential residues were identified which are required for targeting to both cellular compartments. Evidence suggests that mitochondrial Cdc17 function is required for survival of <i>S. pombe</i>. The PCNA binding motif is functional <i>in vivo</i> since its absence reduces cell viability. Despite these identified function overexpression of truncations lacking the N-terminal domain can complement a <i>cdc17</i> deletion strain.

A high-throughput hydrogen peroxide-based colorimetric screen used to detect oxidase activity was extended to detect racemase activity through the production of the specific substrate for an enantioselective oxidase enzyme. A two-plasmid system (encoding the racemase and oxidase) was shown to be successful and could have possible applications for many different classes of enzymes that produce an oxidase substrate. In order to validate the screening technology, three amino acid racemase genes were cloned from genomic DNA and placed into expression vectors. The screen was used to confirm the substrate specificities of each racemase. The amino acid racemases were then subjected to random mutagenesis using the mutator strain method and error-prone polymerase chain reaction. Libraries of the variant racemase enzymes were screened for novel activities towards substrates not accepted by the wild type enzymes: L-arginine; L-lysine and L-leucine. Novel activity was discovered in selected <i>Streptomyces coelicolor </i>alanine racemase variants. Sequencing revealed that the best variant had three point mutations I195T, N223D and I374N. Computer modelling suggested that the I374N mutation was a key mutation and so the variant containing the double mutation (I195T and N223D) was prepared. Partial purification of the wild type enzyme, the variants containing the double and the triple mutations was carried out to compare the substrate specificities. The I195T/N223D variant was shown to be ten times more active towards L-arginine that the wild type enzyme, and the variant containing all three mutations was shown to be 100 times more active towards L-arginine than the wild type enzyme. Both variants displayed novel activity towards L-arginine only.

This thesis describes the design and synthetic approach to a potential transition state inhibitor 26 of an α(1.3)-fucosyltransferase - an important enzyme involved in the inflammatory response where white blood cells are recruited to the site of injury. Fig. 1227A. Chapter 1 reviews the literature of potential inhibitors of both glycosyltransferases and glycosidases and outlines the aim of the project. Chapter 2 reveals the retrosynthetic strategy adopted for the trisubstrate analogue 26 and of the core azasugar portion which is a <I>bis-</I>hydroxymethyl piperidine 27. The synthetic challenge in this project resided in the synthesis of this unit and initially, an asymmetric synthesis of the known azasugar L-deoxyfuconojirimycin (DFJ) 7 which contains four of the required stereogenic centres of the core azasugar 27 was investigated. Chapter 3 describes the successful synthesis of 7 in 14 steps commencing from the readily available amino acid <I>N</I>-Box-L-alanine 51. Fig. 1227B. It was envisaged that the route to DFJ could subsequently be elaborated to the <I>bis</I>-dihydroxymethyl piperidine 27, but due to unforeseen problems a revised approach to the prime target from <I>N</I>-Box-L-alanine 51 was adopted. Chapter 4 outlines 10 steps that have been completed <I>en route</I> to an appropriately functionalised acyclic system which is anticipated to undergo a Lewis acid catalysed <I>6-exo-tet</I> cyclisation of an amine function onto a gem-disubstituted epoxide to furnish a cyclised precursor to the target azasugar 27.