<p>To cope with various endogenous toxin and xenobiotics nature has equipped the organisms with a proper protection system. Glutathione transferases (GSTs) are important components of the cellular defense against oxidative stress. These proteins appear to be suited for different tasks.</p><p>Based on catalytic activity of GSTs with monochlorobimane (MCB), a screening method was developed for identification of active GSTs in bacterial colonies and for characterization of combinatorial GST libraries. </p><p>Solvent viscosity effects on k_cat and k_cat/K_m on wild-type human GST A1-1 and phenylalanine-220 mutants indicate a physical step being the rate-limiting step in the catalytic mechanism.</p><p>Three residues that were under evolutionary selection pressure were identified in Mu class GSTs. By changing these residues in human GSTM2-2, a 1000-fold change of catalytic activity towards GSTM1-1 was accomplished. </p><p>Using peptide phage display, a peptide sequence was found that acts as non-substrate ligand for human GST M2-2. The peptide sequence was shown to be highly similar to the C-terminal region of c-Jun N-terminal kinase (JNK). JNK is a kinase linked to activating protein-1 (AP-1) transcriptional activity, which is part of the regulation of cell proliferation and apoptosis in response to cellular stress. Reporter gene assays in cell lines showed that human GST M2-2 coactivates the transcriptional activity of AP-1. </p><p>GSTs as part of the cellular defense against oxidative stress could be important in inflammatory processes. The distribution of GSTs in the intestine of both mice and human in abnormal inflammatory state was investigated immunohistochemically. Using an experimental mouse model, it was shown that mouse GST A4-4 is markedly induced while, the expression of Mu and Pi class GSTs is reduced in the colon of conventional and germ-free mice with extensive colitis. Moreover, the expression of mouse GST A4-4 was elevated with time when germ-free mice were exposed to normal bacteria flora. In contrast, Mu and Pi class GSTs showed decreased expression in the colon of germ-free mice associated with commensal flora. The Alpha, Mu and Pi class GST levels in mouse colon were increased when germ-free mice received <i>Lactobacillus</i> strain GG.</p><p>The distribution of Alpha, Mu and Pi class GST in the intestinal tissues of patients with Crohn’s disease was investigated using immunohistochemistry. All the three classes were consistently expressed in the intestinal epithelium as well as in macrophage-like cells and smooth muscle tissue. The mucus secreting goblet cells, however, did not express Alpha class GST.</p>

<p>All respiratory pathways, whether aerobic or anaerobic, are based on formation of an electrochemical proton gradient called proton motive force (pmf) that drives ATP formation. Membrane-bound respiratory complexes translocate protons across the membrane from a region of low [H+] and negative electrical potential to a region of high [H+] and positive electrical potential. This establishes a proton gradient and a membrane potential that together form pmf. </p><p>The crystal structures of the respiratory complexes succinate:ubiquinone oxidoreductase (SQR) and formate dehydrogenase-N (Fdh-N) from E. coli, have been solved to 2.6 and 1.6 Å respectively. The structures reveal detailed information about the structure/function relationship of each enzyme as well as demonstrating how pmf is generated. </p><p>The aerobic respiratory complex SQR is a member of both the citric acid cycle and the respiratory chain. It oxidises succinate to fumarate in the cytoplasm and reduces ubiquinone in the membrane. SQR’s contribution to pmf is by reduction of ubiquinone to ubiquinol, which is subsequently used by other members of the respiratory chain to perform proton translocation. The structure of SQR reveals the location and properties of the ubiquinone binding site and helps explain why mutations in this region result in known diseases in higher organisms.</p><p>Fdh-N, a member of a major anaerobic respiratory pathway in E. coli, forms a redox loop with dissimilatory nitrate reductase, linked by the menaquinone/menaquinol pool. This redox loop contributes to pmf by translocating two protons from the negative to the positive side and two electrons in the opposite direction. The structure of Fdh-N shows the molecular basis of this process and allows for the proposal of a quinone reduction mechanism.</p>

Glutathione transferases (GSTs) make up a superfamily that is involved in the cellular defense against various reactive compounds by catalyzing the conjugation of glutathione to electrophilic centra. Members of this family have also been implicated in different facets of biological signaling. The gene encoding human GST P1-1 is polymorphic, resulting in variant amino acid residues in positions 105 and 114. The role of the polymorphism in the active-site residue 105 on enzyme stability and activity with various substrates was investigated. A valine instead of an isoleucine in position 105 decreased the thermal stability of the enzyme. The effect on enzyme activity was dependent on the substrate and reaction studied. With some substrates tested, such as carcinogenic diolepoxides derived from polyaromatic hydrocarbons, GST P1-1/Val105 displayed the highest catalytic efficiency. In contrast, with 1-chloro-2,4-dinitrobenzene, the GST P1-1/Ile105 showed higher activity. Residue 105 was mutated to alanine and tryptophan to investigate the role of size and hydrophobicity of residue 105 on enzyme properties. Generally, a smaller amino acid in position 105 gave increased activity with large substrates. Clearly, residue 105 of GST P1-1 helps to determine the substrate selectivity of the enzyme. In addition, more voluminous amino acids in position 105 increase the thermal stability of the enzyme. GST P1-1 is believed to contribute to the development of drug resistance in cancer cells. The affinity of GST P1-1 for TER 117, designed to inhibit GST P1-1 in tumors, was not affected by the variability in position 105. TER 117 was found to be a potent inhibitor of glyoxalase I as well. The cDNA encoding GST A3-3 was isolated from a placental cDNA library. GST A3-3 was heterologously expressed, purified and found to catalyze efficiently the double-bond isomerization of Δ5-androstene-3,17-dione and Δ5-pregnene-3,20-dione, reactions taking place in the biosynthesis of the steroid hormones testosterone and progesterone, respectively. GST A3-3 was found to be selectively expressed in steroidogenic tissues, suggesting that this enzyme is involved in the production of steroid hormones. The presence of both the hydroxyl group of the active-site tyrosine 9 and the thiolate form of glutathione, acting as a cofactor, is important for high double-bond isomerase activity. A leucine in position 111 appears to have a major role in productive binding of the steroid substrate but also residues F10 and A216 are determinants for the high isomerase activity. GST A2-2 is a poor catalyst of the steroid double-bond isomerization of Δ5-androstene-3,17-dione as compared to GST A3-3, despite 88% sequence identity. GST A2-2 was redesigned to a highly efficient double-bond isomerase by mutating five active-site residues to the corresponding residues of GST A3-3. This demonstrates the functional plasticity of GSTs and the power of a rational approach to redesign of these enzymes.

Hyaluronan, a component of the extracellular matrix, is synthesized by either of three hyaluronan-synthesizing enzymes termed Has1, Has2 and Has3. The expression level of each Has gene varies between cell types of mesenchymal origin and is differentially regulated in response to external stimuli. For example, stimulation of mesothelial cells with PDGF-BB induced an up-regulation of the Has2 gene, whereas the Has1 and Has3 genes remained unaffected. The induction of Has2 gene expression correlated well with increased Has2 protein levels and accumulation of hyaluronan. Moreover, treatment of mesothelial cells with hydrocortisone suppressed hyaluronan synthesis in cell culture primarily through down-regulation of the Has2 gene. Thus, among the Has isoforms, Has2 seems to be most markedly regulated in response to external stimuli. In an attempt to investigate the importance of hyaluronan in tumor progression, the hyaluronan synthesizing enzyme Has2 and the hyaluronan degrading enzyme Hyal1 were over-expressed in a rat colon adenocarcinoma cell line, PROb. We found that Has2 gene over-expression in colon carcinoma cells promoted cell growth in vitro and progression of transplantable tumors. In contrast, over-expression of Hyal1 lead to a considerable reduction of growth rates both in vivo and in vitro. A linear correlation between tumor growth rate and hyaluronan amount in tumor tissue was observed. In another tumor model, experimental anaplastic thyroid carcinoma, the effects of TGF-β inhibition on hyaluronan and collagen contents in tumor xenografts were investigated. We found that inhibition of TGF-β, a stimulator of hyaluronan and collagen synthesis, lead to reduced collagen deposition whereas the hyaluronan levels in stromal tissue only marginally differed. Our results indicate that a high ratio of collagen to hyaluronan may be characteristic of a pathogenic mechanism that leads to elevated interstitual tumor pressure.

To cope with various endogenous toxin and xenobiotics nature has equipped the organisms with a proper protection system. Glutathione transferases (GSTs) are important components of the cellular defense against oxidative stress. These proteins appear to be suited for different tasks. Based on catalytic activity of GSTs with monochlorobimane (MCB), a screening method was developed for identification of active GSTs in bacterial colonies and for characterization of combinatorial GST libraries. Solvent viscosity effects on kcat and kcat/Km on wild-type human GST A1-1 and phenylalanine-220 mutants indicate a physical step being the rate-limiting step in the catalytic mechanism. Three residues that were under evolutionary selection pressure were identified in Mu class GSTs. By changing these residues in human GSTM2-2, a 1000-fold change of catalytic activity towards GSTM1-1 was accomplished. Using peptide phage display, a peptide sequence was found that acts as non-substrate ligand for human GST M2-2. The peptide sequence was shown to be highly similar to the C-terminal region of c-Jun N-terminal kinase (JNK). JNK is a kinase linked to activating protein-1 (AP-1) transcriptional activity, which is part of the regulation of cell proliferation and apoptosis in response to cellular stress. Reporter gene assays in cell lines showed that human GST M2-2 coactivates the transcriptional activity of AP-1. GSTs as part of the cellular defense against oxidative stress could be important in inflammatory processes. The distribution of GSTs in the intestine of both mice and human in abnormal inflammatory state was investigated immunohistochemically. Using an experimental mouse model, it was shown that mouse GST A4-4 is markedly induced while, the expression of Mu and Pi class GSTs is reduced in the colon of conventional and germ-free mice with extensive colitis. Moreover, the expression of mouse GST A4-4 was elevated with time when germ-free mice were exposed to normal bacteria flora. In contrast, Mu and Pi class GSTs showed decreased expression in the colon of germ-free mice associated with commensal flora. The Alpha, Mu and Pi class GST levels in mouse colon were increased when germ-free mice received Lactobacillus strain GG. The distribution of Alpha, Mu and Pi class GST in the intestinal tissues of patients with Crohn’s disease was investigated using immunohistochemistry. All the three classes were consistently expressed in the intestinal epithelium as well as in macrophage-like cells and smooth muscle tissue. The mucus secreting goblet cells, however, did not express Alpha class GST.

All respiratory pathways, whether aerobic or anaerobic, are based on formation of an electrochemical proton gradient called proton motive force (pmf) that drives ATP formation. Membrane-bound respiratory complexes translocate protons across the membrane from a region of low [H+] and negative electrical potential to a region of high [H+] and positive electrical potential. This establishes a proton gradient and a membrane potential that together form pmf. The crystal structures of the respiratory complexes succinate:ubiquinone oxidoreductase (SQR) and formate dehydrogenase-N (Fdh-N) from E. coli, have been solved to 2.6 and 1.6 Å respectively. The structures reveal detailed information about the structure/function relationship of each enzyme as well as demonstrating how pmf is generated. The aerobic respiratory complex SQR is a member of both the citric acid cycle and the respiratory chain. It oxidises succinate to fumarate in the cytoplasm and reduces ubiquinone in the membrane. SQR’s contribution to pmf is by reduction of ubiquinone to ubiquinol, which is subsequently used by other members of the respiratory chain to perform proton translocation. The structure of SQR reveals the location and properties of the ubiquinone binding site and helps explain why mutations in this region result in known diseases in higher organisms. Fdh-N, a member of a major anaerobic respiratory pathway in E. coli, forms a redox loop with dissimilatory nitrate reductase, linked by the menaquinone/menaquinol pool. This redox loop contributes to pmf by translocating two protons from the negative to the positive side and two electrons in the opposite direction. The structure of Fdh-N shows the molecular basis of this process and allows for the proposal of a quinone reduction mechanism.

<p>The goal of this work was to synthesize different dendritic architectures and evaluate the effect from the dendrons on the material properties. The work presented in this licentiate thesis, Synthesis and Characterization of Dendritic Architectures, is divided into major parts. The first part deals with the synthesis and characterization of two sets of dendritic porphyrins based on 2,2-bis(methylol)propionic acid (bis-MPA). The second part deals with the synthesis and characterization of a series of dendronized polymers based on bis-MPA.</p><p>Both free-base and zinc containing dendritic porphyrins were synthesized up to the fifth generation utilizing the acetonide protected anhydride of bis-MPA. The resulting dendrimers were characterized by SEC, NMR, and MALDI-TOF. The dendrimers were found to be well-defined, virtually monodisperse, molecules up to the fourth generation. In the case of the fifth generation dendrimers, some structural defects were observed. The hydrodynamic volume (in THF) of these molecules was calculated using the rotational correlation time, and they were found to be more compact than the corresponding Fréchet-type dendrimers of the same generation.</p><p>Macromonomers of the first and second generation were also synthesized utilizing the acetonide protected anhydride of bis-MPA and subsequently polymerized by atom transfer radical polymerization, using a system of N-propyl-2-pyridylmethanamine, Cu(I)Br, and Cu(I)Br2. This system resulted in well-controlled polymerizations with low polydispersity polymers. By adopting a divergent ‘graft-to’ approach, welldefined dendronized polymers with acetonide, hydroxyl, acetate, and hexadecyl functionality respectively, were obtained.</p><p>The bulk properties of the dendronized polymers were investigated by differential scanning calorimetry, dynamic-mechanical measurements, and 1H-NMR selfdiffusion. It was found that that increasing the size of the pendant dendron increased the glass transition temperature of the materials. The degree of crystallization of the hexadecyl functional materials was found to decrease with dendron size, most likely due to the reduced flexibility of the backbone prohibiting effective crystallization. The dynamic mechanical measurements revealed that the behavior of the complex viscosity as a function of frequency was independent of functionality. The second and third generation materials were found to have a Newtonian plateau up to a frequency where they become shear-thinning. The fourth generation materials were found to be shearthinning in the frequency range. 1H-NMR self-diffusion measurements revealed that the shape of the acetonide functional dendronized polymers in solution was best described by using a rod-like or prolated ellipsoid model.</p>

<p>The present thesis concerns theoretical studies of molecular interactions investigated by infrared and X-ray spectroscopic techniques, with emphasis on using the two technologies combined in pump probe experiments. Three main types of studies are addressed: the use of near-edge X-ray absorption fine structure spectra (NEXAFS) to manifest through-bond and through-space interactions; the role of hydrogen bonding on the formation of X-ray photoelectron spectra as evidenced by simulations of the water dimer; and the development of theory, with sample applications, for infrared X-ray pump probe spectroscopy - the main theme of the thesis.</p><p>Ab initio calculations indicate that NEXAFS spectra give direct information about the through-bond and through-space interactions between vacant non-conjugated π* orbitals. It is found that the X-ray photoelectron spectrum of the water dimer differs strongly from the monomer spectrum in that two bands are observed, separated by the chemically shifted ionization potentials of the donor and the acceptor. The hydrogen bond is responsible for the anomalously strong broadening of these two bands. The studies show that X-ray core electron ionization of the water dimer driven by an infrared field is a proper technique to prove the proton transfered state contrary to conventional X-ray photoelectron spectroscopy. Our simulations of infrared X-ray pump-probe spectra were carried out using wave packet propagation techniques.</p><p>The physical aspects of the proposed new X-ray spectroscopic method - phase sensitive Infrared - X-ray pump probe spectroscopy - are examined in detail in two sample applications - on the NO molecule and on the dynamics of proton transfer in core ionized water dimer. It is found that the phase of the infrared pump field strongly influences the trajectory of the nuclear wave packet on the ground state potential. This results in a phase dependence of the X-ray pump probe spectra. A proper choice of the delay time of the X-ray pulse allows to directly observe the X-ray transition in the proton transfered well of the core excited potential.</p>

The present work focuses on the glutathione transferase (GST) Alpha-class enzymes, their characteristics as steroid isomerases and structural plasticity as malleable scaffolds for protein design. The GSTs are a family of detoxication enzymes that appears to have a wider variety of additional functions. Kinetic steady-state parameters for human GST A1-1 with the steroid isomerase substrate Δ5-androstene-3,17-dione (AD), an intermediate in steroid hormone biosynthesis, were determined. It was established that GST A1-1 is a highly efficient steroid isomerase with a 30-fold higher catalytic efficiency, in terms of kcat/Km, than 3β-hydroxysteroid dehydrogenase/Δ5→4-isomerase, the enzyme regarded as the mammalian Δ5→4-isomerase in steroid hormone biosynthesis. Kinetic parameters were also determined for GST A2-2, GST A4-4 and the GST A1-1 mutant Y9F. From the dependency on pH of the kinetic parameters it was established that efficient catalysis requires glutathione (GSH) in its deprotonated form and it is suggested that the GSH-thiolate acts as a base in the catalysis of the Δ5→4-3-ketosteroid isomerase reaction. GST A2-2 is a poor catalyst of the steroid isomerase reaction while GST A3-3 is highly efficient. Their catalytic efficiencies (kcat/Km) differ 5000-fold. Stepwise point mutations were performed to GST A2-2 in order to insert the amino acid residues from the active-site of GST A3-3 that distinguishes the two isoenzymes. The result was that GST A2-2 was redesigned to a highly efficient double-bond isomerase with both the catalytic constant (kcat) and catalytic efficiency (kcat/Km) in the same order as for GST A3-3. Furthermore, this was done by only exchanging amino-acid residues with first-sphere interactions, providing empirical proof-of principle for knowledge-based enzyme design. Kinetic studies on GST A1-1 and a T68E mutant of GST A1-1 were also performed with a GSH analog lacking the g-glutamate a-carboxylate (dGSH), and using three different electrophilic substrates (AD; 1-chloro-2,4-dinitrobenzene, CDNB; 4-nitrocinnamaldehyde). Deletion of the a-carboxylate from the GSH glutamate had a severe impact on all reaction constants and it changed the rate-limiting step for the CDNB reaction as well as changed the pKa value for the enzyme-bound GSH thiol. The loss in activity caused by dGSH could in part be compensated by the T68E mutant contributing an enzyme-bound carboxylate instead. The C-terminus of GST A1-1 is flexible and folds over the active site when the enzyme binds a substrate. Phenylalanine residues in the C-terminal end, known to interact with active-site residues tyrosine 9 and phenylalanine 10, were mutated to abolish those interactions. Studies of viscosity dependence for CDNB and AD with regard to kcat and kcat/Km showed that the dynamic C-terminal segment influence rate-determining steps for both the larger isomerase substrate, AD, as well as for the smaller conjugation substrate, CDNB.

<p>A microorganism was isolated which could grow on unusually high concentrations of the toxic pollutant 4-chlorophenol. Taxonomic studies showed that the microorganism constituted a novel species within the genus <i>Arthrobacter</i> and it was named <i>Arthrobacter chlorophenolicus</i> A6. </p><p><i>A. chlorophenolicus</i> A6 was chromosomally tagged with either the <i>gfp</i> gene, encoding the green fluorescent protein (GFP), or the <i>luc</i> gene, encoding firefly luciferase. When the tagged cells were inoculated into 4-chlorophenol contaminated soil they could completely remove 175 µg/g 4-chlorophenol within 10 days, whereas no loss of 4-chlorophenol was observed in the uninoculated control microcosms. During these experiments the <i>gfp</i> and <i>luc</i> marker genes allowed monitoring of cell number and metabolic status.</p><p>When <i>A. chlorophenolicus</i> A6 was grown on mixtures of phenolic compounds, the strain exhibited a preference for 4-nitrophenol over 4-chlorophenol, which in turn was preferred over phenol. Analysis of growth and degradation data indicated that the same enzyme system was used for removal of 4-chlorophenol and 4-nitrophenol. However, degradation of unbstituted phenol appeared to be mediated by another or an additional enzyme system. The <i>luc</i>-tagged <i>A. chlorophenolicus</i> A6 gave valuable information about growth, substrate depletion and toxicity of the phenolic compounds in substrate mixtures. </p><p>The 4-chlorophenol degradation pathway in <i>A. chlorophenolicus</i> A6 was elucidated. The metabolic intermediate subject to ring cleavage was found to be hydroxyquinol and two different pathway branches led from 4-chlorophenol to hydroxyquinol. A gene cluster involved in 4-chlorophenol degradation was cloned from <i>A. chlorophenolicus A6</i>. The cluster contained two functional hydroxyquinol 1,2-dioxygenase genes and a number of other open reading frames presumed to encode enzymes involved in 4-chlorophenol catabolism. Analysis of the DNA sequence suggested that the gene cluster had partly been assembled by horizontal gene transfer.</p><p>In summary, 4-chlorophenol degradation by <i>A. chlorophenolicus</i> A6 was studied from a number of angles. This organism has several interesting and useful traits such as the ability to degrade high concentrations of 4-chlorophenol and other phenols alone and in mixtures, an unusual and effective 4-chlorophenol degradation pathway and demonstrated ability to remove 4-chlorophenol from contaminated soil.</p>