<p>In the pentose phosphate pathway, carbohydrates such as glucose and ribose are degraded with production of reductive power and energy. Another important function is to produce essential pentoses, such as ribose 5-phosphate, which later can be used in biosynthesis of nucleic acids and cofactors. </p><p>This thesis presents structural and functional studies on three enzymes involved in ribose metabolism in <i>Escherichia coli</i>. </p><p>Ribokinase is an enzyme that phosphorylates ribose in the presence of ATP and magnesium, as the first step of exogenous ribose metabolism. Two important aspects of ribokinase function, not previously known, have been elucidated. Ribokinase was shown to be activated by monovalent cations, specifically potassium. Structural analysis of the monovalent ion binding site indicates that the ion has a structural rather than catalytic role; a mode of activation involving a conformational change has been suggested. Product inhibition studies suggest that ATP is the first substrate to bind the enzyme. Independent K_d measurements with the ATP analogue AMP-PCP support this. The results presented here will have implications for several enzymes in the protein family to which ribokinase belongs, in particular the medically interesting enzyme adenosine kinase. </p><p>Ribose 5-phosphate isomerases convert ribose 5-phosphate into ribulose 5-phosphate or <i>vice versa</i>. Structural studies on the two genetically distinct isomerases in <i>E. coli</i> have shown them to be fundamentally different in many aspects, including active site architecture. However, a kinetic study has demonstrated both enzymes to be efficient in terms of catalysis. Sequence searches of completed genomes show ribose 5-phosphate isomerase B to be the sole isomerase in many bacteria, although ribose 5-phosphate isomerase A is a nearly universal enzyme. All genomes contain at least one of the two enzymes. These results confirm that both enzymes must be independently capable of supporting ribose metabolism, a fact that had not previously been established.</p>

<p>Polyunsaturated fatty acids can be bioactivated by two families of dioxygenases, which either contain non-heme iron (lipoxygenases) or heme (cyclooxygenases, linoleate diol synthases and α-dioxygenases).</p><p>Lipoxygenases and their products play important roles in the pathophysiology of plants and fungi. The only known lipoxygenase with catalytic manganese (Mn-lipoxygenase) is secreted by a devastating root pathogen of wheat, the Take-all fungus <i>Gaeumannomyces graminis</i>. Its mycelia also contains linoleate diol synthase (LDS), which can oxidize linoleic acid to sporulation hormones.</p><p>Mn-lipoxygenase belongs to the lipoxygenase gene family. Recombinant Mn-lipoxygenase was successfully expressed in the yeast <i>Pichia pastoris</i> with an expression level of 30 mg/L in fermentor culture. The tentative metal ligands of Mn-lipoxygenase were studied by site-directed mutagenesis. The results show that four residues His-274, His-278, His-462 and the C-terminal Val-602 likely coordinate manganese, as predicted by sequence alignments with Fe lipoxygenases.</p><p>Mn-lipoxygenase (~100 kDa) contains an Asp-Pro peptide bond in the N-terminal region, which appears to hydrolyze during storage and in the acidic media during Pichia expression to an active enzyme of smaller size, mini-Mn-lipoxygenase (~70 kDa). The active form of Mn-lipoxygenase can oxygenate fatty acids of variable chain length, suggesting that the fatty acids enter the catalytic site with the ω-end (“tail first”).</p><p>Mn-lipoxygenase is an <i>R</i>-lipoxygenase with a conserved Gly316 residue known as a determinant of stereospecificity in other <i>R/S</i> lipoxygenases. The Gly316Ala mutant showed an increased hydroperoxide isomerase activity and transformed 18:3n-3 and 17:3n-3 to epoxyalcohols.</p><p>The genome of the rice blast fungus, <i>Magnaporthe grisea</i>, contains putative genes of lipoxygenases and LDS. Mycelia of <i>M. grisea</i> were found to express LDS activity. This enzyme was cloned and sequenced and showed 65% amino acid identity with LDS from <i>G.graminis</i>. </p><p>Take-all and the rice blast fungi represent a constant threat to staple foods worldwide. Mn-lipoxygenase and LDS might provide new means to combat these pathogens.</p>

<p>Ribose 5-phosphate isomerase (Rpi) is one of the major enzymes of the pentose phosphate pathway, where it catalyses the inter-conversion of ribose 5-phosphate (R5P) and ribulose 5-phosphate. Two forms of this isomerase with no significant amino acid sequence similarity exist, RpiA and RpiB. This thesis describes RpiB from the organisms <i>Mycobacterium tuberculosis</i> (<i>Mt</i>) and <i>Escherichia coli</i> (<i>Ec</i>) from a structural and functional point of view.</p><p>Since the <i>E. coli</i> genome encodes both an RpiA and an RpiB, which generally is not expressed, it has been proposed that <i>Ec</i>RpiB has a different role as an allose-6-phosphate isomerase. Activity measurements presented here show that <i>Ec</i>RpiB does have this second activity. </p><p>In the <i>M. tuberculosis</i> genome there is only a gene for RpiB. The crystal structure of <i>Mt</i>RpiB was solved in complex with several different inhibitors designed to mimic the reaction intermediate as well as with the substrate, R5P. The organisation of the active site in these structures could be used to derive the reaction mechanism for <i>Mt</i>RpiB and for other RpiBs in general. Activity measurements of <i>Mt</i>RpiB showed that it can catalyse the R5P isomerisation, but not the allose 6-phosphate reaction. Differences observed in the active site between <i>Ec</i>RpiB and <i>Mt</i>RpiB explain these kinetic results. </p><p>Activity measurements and a structure of an <i>Ec</i>RpiB mutant, where histidine99 was changed to asparagine, implies that RpiB catalyses the first step of the reaction in which the sugar ring must be opened, and gives a possible explanation for how this could occur. </p><p>Inhibition studies have uncovered a compound that selectively inhibits <i>Mt</i>RpiB over RpiA from spinach, which is homologous to the human RpiA. Differences in the inhibition patterns and active site residues of these two species’ Rpi may provide information for future virtual screening approaches, with the aim of discovering new anti-tuberculosis agents.</p>

In the pentose phosphate pathway, carbohydrates such as glucose and ribose are degraded with production of reductive power and energy. Another important function is to produce essential pentoses, such as ribose 5-phosphate, which later can be used in biosynthesis of nucleic acids and cofactors. This thesis presents structural and functional studies on three enzymes involved in ribose metabolism in Escherichia coli. Ribokinase is an enzyme that phosphorylates ribose in the presence of ATP and magnesium, as the first step of exogenous ribose metabolism. Two important aspects of ribokinase function, not previously known, have been elucidated. Ribokinase was shown to be activated by monovalent cations, specifically potassium. Structural analysis of the monovalent ion binding site indicates that the ion has a structural rather than catalytic role; a mode of activation involving a conformational change has been suggested. Product inhibition studies suggest that ATP is the first substrate to bind the enzyme. Independent Kd measurements with the ATP analogue AMP-PCP support this. The results presented here will have implications for several enzymes in the protein family to which ribokinase belongs, in particular the medically interesting enzyme adenosine kinase. Ribose 5-phosphate isomerases convert ribose 5-phosphate into ribulose 5-phosphate or vice versa. Structural studies on the two genetically distinct isomerases in E. coli have shown them to be fundamentally different in many aspects, including active site architecture. However, a kinetic study has demonstrated both enzymes to be efficient in terms of catalysis. Sequence searches of completed genomes show ribose 5-phosphate isomerase B to be the sole isomerase in many bacteria, although ribose 5-phosphate isomerase A is a nearly universal enzyme. All genomes contain at least one of the two enzymes. These results confirm that both enzymes must be independently capable of supporting ribose metabolism, a fact that had not previously been established.

Polyunsaturated fatty acids can be bioactivated by two families of dioxygenases, which either contain non-heme iron (lipoxygenases) or heme (cyclooxygenases, linoleate diol synthases and α-dioxygenases). Lipoxygenases and their products play important roles in the pathophysiology of plants and fungi. The only known lipoxygenase with catalytic manganese (Mn-lipoxygenase) is secreted by a devastating root pathogen of wheat, the Take-all fungus Gaeumannomyces graminis. Its mycelia also contains linoleate diol synthase (LDS), which can oxidize linoleic acid to sporulation hormones. Mn-lipoxygenase belongs to the lipoxygenase gene family. Recombinant Mn-lipoxygenase was successfully expressed in the yeast Pichia pastoris with an expression level of 30 mg/L in fermentor culture. The tentative metal ligands of Mn-lipoxygenase were studied by site-directed mutagenesis. The results show that four residues His-274, His-278, His-462 and the C-terminal Val-602 likely coordinate manganese, as predicted by sequence alignments with Fe lipoxygenases. Mn-lipoxygenase (~100 kDa) contains an Asp-Pro peptide bond in the N-terminal region, which appears to hydrolyze during storage and in the acidic media during Pichia expression to an active enzyme of smaller size, mini-Mn-lipoxygenase (~70 kDa). The active form of Mn-lipoxygenase can oxygenate fatty acids of variable chain length, suggesting that the fatty acids enter the catalytic site with the ω-end (“tail first”). Mn-lipoxygenase is an R-lipoxygenase with a conserved Gly316 residue known as a determinant of stereospecificity in other R/S lipoxygenases. The Gly316Ala mutant showed an increased hydroperoxide isomerase activity and transformed 18:3n-3 and 17:3n-3 to epoxyalcohols. The genome of the rice blast fungus, Magnaporthe grisea, contains putative genes of lipoxygenases and LDS. Mycelia of M. grisea were found to express LDS activity. This enzyme was cloned and sequenced and showed 65% amino acid identity with LDS from G.graminis. Take-all and the rice blast fungi represent a constant threat to staple foods worldwide. Mn-lipoxygenase and LDS might provide new means to combat these pathogens.

Ribose 5-phosphate isomerase (Rpi) is one of the major enzymes of the pentose phosphate pathway, where it catalyses the inter-conversion of ribose 5-phosphate (R5P) and ribulose 5-phosphate. Two forms of this isomerase with no significant amino acid sequence similarity exist, RpiA and RpiB. This thesis describes RpiB from the organisms Mycobacterium tuberculosis (Mt) and Escherichia coli (Ec) from a structural and functional point of view. Since the E. coli genome encodes both an RpiA and an RpiB, which generally is not expressed, it has been proposed that EcRpiB has a different role as an allose-6-phosphate isomerase. Activity measurements presented here show that EcRpiB does have this second activity. In the M. tuberculosis genome there is only a gene for RpiB. The crystal structure of MtRpiB was solved in complex with several different inhibitors designed to mimic the reaction intermediate as well as with the substrate, R5P. The organisation of the active site in these structures could be used to derive the reaction mechanism for MtRpiB and for other RpiBs in general. Activity measurements of MtRpiB showed that it can catalyse the R5P isomerisation, but not the allose 6-phosphate reaction. Differences observed in the active site between EcRpiB and MtRpiB explain these kinetic results. Activity measurements and a structure of an EcRpiB mutant, where histidine99 was changed to asparagine, implies that RpiB catalyses the first step of the reaction in which the sugar ring must be opened, and gives a possible explanation for how this could occur. Inhibition studies have uncovered a compound that selectively inhibits MtRpiB over RpiA from spinach, which is homologous to the human RpiA. Differences in the inhibition patterns and active site residues of these two species’ Rpi may provide information for future virtual screening approaches, with the aim of discovering new anti-tuberculosis agents.

Mycobacterium tuberculosis, the causative pathogen of tuberculosis, currently infects one-third of the world’s population, resulting in two million deaths annually. This clearly shows that tuberculosis is one of the most serious diseases of our times. The often unpleasant side effects from the current drugs, combined with the difficulty of ensuring patient compliance, and the emergence of drug-resistant and multidrug-resistant strains, makes the need for new and better drugs urgent. In this thesis, all the steps, from cloning, purification, crystallization, to activity determination, and structure determination are presented for two different M. tuberculosis enzymes. The structures, which were modeled from X-ray crystallographic data, provide the framework for structure-based drug design. Here, new potential inhibitors can be tailor-made based on the specific interactions in the enzyme’s active site. The bacteria have two different peptidyl-prolyl cis-trans isomerases that catalyze the isomerization of peptide bonds preceding proline residues, a process of high importance for correct folding. Here we present the structure of peptidyl-prolyl cis-trans isomerase A, an enzyme present inside the bacteria, and distinguish it from the B form of the enzyme, which is membrane bound, placing its active site outside the bacteria. The enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase catalyzes the second step within the non-mevalonate pathway, which leads to the production of isopentenyl diphosphate. This compound is the precursor of various isoprenoids, vital to all living organisms. In humans, isopentenyl diphosphate is produced via a different pathway, indicating that all the enzymes within the non-mevalonate pathway may be suitable drug targets in M. tuberculosis. Several structures of both wild type and mutant 1-deoxy-D-xylulose-5-phosphate reductoisomerase in complex with different substrates, and also with the known inhibitor fosmidomycin, provide valuable information not only to the field of drug design, but also, in this case, into the catalysis.

Many members of the Phylum Cnidaria are mutualistic with unicellular dinoflagellates belonging to the genus Symbiodinium. Corals are the most widely recognized example of these associations due to their key ecological importance in coral reef ecosystems where they serve as the structural and trophic foundation of these rich ecosystems. Coral reefs are severely threatened by human activities worldwide and are at great risk from global climate change, in particular the increase in seasurface temperatures. Detailed knowledge of how corals respond to stress is scarce. The most serious and immediate response of corals to environmental stress is a process referred to as coral bleaching (a.k.a. cnidarian bleaching). Nevertheless, the cellular and molecular processes by which elevated temperatures elicit the bleaching response are poorly understood. This dissertation deals with this important question by describing two mediators of cnidarian bleaching in the model symbiotic tropical sea anemone Aiptasia pallida (Verril), namely nitric oxide and cyclophilin. After an introduction to the topic of cnidarian-algal symbioses and cnidarian bleaching (Chapter 1), I present results from a study describing the involvement of nitric oxide (NO) in the anemone A. pallida (Chapter 2). Elevated temperature as well as oxidative stress induces production of NO and exposure of A. pallida to NO induces bleaching at non-stressful temperatures. Co-incubation with an NO scavenger suppresses bleaching. I propose that the host up-regulates NO production in response to elevated oxidative stress and that this situation leads to cytotoxicity and bleaching. Chapter 3 examines the role of cyclophilin from A. pallida in the regulation of the symbiosis. Cyclophilins belong to a highly conserved family peptydyl-prolyl cistrans isomerases (PPIases). Incubation of A. pallida with cyclosporin A (CsA), a potent inhibitor of cyclophilin resulted in bleaching and a decrease in tolerance to elevated temperatures. Protein extracts from A. pallida exhibited CsA-sensitive PPIase activity. Laser scanning confocal microscopy using superoxide and nitric oxide-sensitive fluorescent dyes on live A. pallida revealed that CsA strongly induced the production reactive oxygen species as well as NO. We tested weather the CsAsensitive isomerase activity is important for maintaining the activity of the antioxidant enzyme superoxide dismutase (SOD). SOD activity of protein extracts was not affected by pre-incubation with CsA in vitro. In Chapter 4 I review what is known about the molecular and cellular mechanisms of bleaching and describe a model of bleaching based on the results presented herein as well as studies of non-cnidarian models. / Graduation date: 2007

In this study, process development for glucose isomerase (GI) was aimed. In this context, firstly, thermostable xyl genes, PCR amplified from Thermus thermophilus and Pyrococcus furiosus cells, were recombined to the E.coli BL21 (DE3) and P.pastoris strains, respectively. But significant increase in the term of GI activity compared with wild type cells only detected in recombinant E.coli strain so this strain was selected for further experiments. Then, the effect of different natural and artificial inducers on the production of rGI under control of LacUV5 promoter was investigated in laboratory-scale bioreactors. Lactose was shown to be more efficient in the term of operon induction for long time bioprocesses. Thereafter, in order to increase thermostable rGI production rate, to achieve high cell density culture of E.coli BL21 (DE3) pLysS pRSETA::xylT as well as to evade acetate accumulation, the effect of exponential feeding strategy of carbon source on the production of thermostable GI enzyme, cell concentration and acetate formation by recombinant E.coli BL21 (DE3) pLysS was investigated at four sets of fed-batch bioreactor experiments at three different predetermined specific growth rates 0.1 h-1 (M-0.1), 0.15 h-1 (M-0.15), 0.2 h-1 (M-0.2) and a glucose based exponential feeding at specific growth rate of 0.15 h-1(G-0.15) were performed by recombinant E.coli BL21 (DE3) pLysS cells. The highest biomass was obtained in M-0.15 condition as 9.6 kg m&minus / 3 at t=32 h and the highest rGI activity was achieved in M-0.1 operation as A=16399 U L-1 at t=32 h of bioprocess. Moreover, peptide ligand with specific affinity toward histidin-tag peptide was selected by phage display technology. Isothermal titration calorimetry and surface plasmon resonance analyses were carried out to determine peptide-peptide interaction properties.

This thesis describes studies directed towards understanding structure-function relationships of triosephosphate isomerase (TIM), from a protozoan parasite Plasmodium falciparum and a thermophilic archaea Methanocaldococcus jannaschii. Triosephosphate isomerase, a ubiquitous glycolytic enzyme, has been the subject of biochemical, enzymatic and structural studies for the last five decades. Studies on TIM have been central to the development of mechanistic enzymology. The present study investigates the role of specific residues in the structure and function of Plasmodium falciparum triosephosphate isomerase (PfTIM). The structure and stability of a tetrameric triosephosphate isomerase from Methanocaldococcus jannaschii (MjTIM) is also presented. Chapter 1 provides a general introduction to the glycolytic enzyme triosephosphate isomerase, conservation of TIM sequences, its fold and three dimensional organization. The isomerisation reaction interconverting dihydroxyacetone phosphate and glyceraldehyde 3phosphate catalyzed by triosephosphate isomerase is an example of a highly stereospecific proton transfer process (Hall & Knowles, 1975; Rieder & Rose, 1959). This chapter briefly reviews mechanistic features and discusses the role of active site residues and the functional flexible loop 6. Triosephosphate isomerase adopts the widely occurring ( β/ α)8 barrel fold and mostly occurs as a dimer (Banner et al., 1975). Protein engineering studies, related to folding, stability and design of monomeric TIM are also addressed. A brief introduction to thermophilic TIMs and higher oligomeric TIMs is given. The role of this enzyme in disease states like hemolytic anemia and neuromuscular dysfunction is surveyed. The production of methylglyoxal, a toxic metabolite, as a byproduct of the TIM reaction is also considered. Many proteins utilize segmental motions to catalyze a specific reaction. The omega loop (loop 6) of triosephosphate isomerase is important for preventing the ene-diol intermediate from forming the cytotoxic byproduct, methylglyoxal. The active site loop-6 of triosephosphate isomerase moves about 7Ǻ on ligand binding. It exhibits a hinged lid motion alternating between two well defined, “open” and “closed”, conformations (Joseph et al., 1990). Though the movement of loop 6 is not ligand gated, in crystals the ligand bound forms invariably reveal a closed loop conformation. Plasmodium falciparum TIM is an exception which predominantly exhibits “open” loop conformations, even in the ligand bound state (Parthasarathy et al., 2002). Phe 96 is a key residue that is involved in contacts between the flexible loop-6 and the protein body in PfTIM. Notably, in all TIM sequences determined thus far, with the exception of plasmodial sequences, this residue is Ser 96. In Chapter 2 the mutants F96S, F96H and F96W are reported. The crystal structures of the mutant enzymes with or without bound ligand are described. In all the ligand free cases, loop-6 adopts an “open” conformation. Kinetic parameters for all the mutants establish that residue 96 does not play an essential role in modulating the loop conformation but may be important for ligand binding. Structural analysis of the mutants along with WT enzyme reveals the presence of a water network which can modulate ligand binding. Subunit interfaces of oligomeric proteins provide an opportunity to understand protein- protein interactions. Chapter 3 describes biochemical and biophysical studies on two separate dimer-interface destabilizing mutants C13E and W11F/W168F/Y74W of PfTIM. The intention was to generate a stable monomer by disrupting the interaction hubs. C13 is a part of a large hydrophobic patch (Maithal et al., 2002a) at the dimer interface. Introduction of a negative charge at position 13 destabilizes the interface and reduces activity. Y74 is a part of an aromatic cluster of the interface (Maithal et al., 2002b). The Y74W triple mutant was designed to disrupt the aromatic cluster by introducing additional atoms. Tryptophan is also a fluorophore, allowing studies of the dimer disruption by fluorescence, after mutating the two inherent tryptophan residues, W11 and W168 to phenylalanine. The mutants showed reduced activity and were more sensitive than the wild type enzyme to chemical denaturants as well as thermal denaturation. Evidenced for monomer formation is presented. These studies together with previous work reveal that the interface is important for both activity and stability. In order to develop a model for understanding the relationship between protein stabilization and oligomeric status, characterization of the TIM from Methanocaldococcus jannaschii (MjTIM) has been undertaken. Chapter 4 describes the purification and characterization of MjTIM. The MjTIM gene was cloned and expressed in pTrc99A and protein was isolated from AA200 E. coli cells. Hyperexpressed protein was purified to homogeneity and relevant kinetic parameters have been determined. The tetrameric nature of MjTIM is established by gel filtration studies. Circular dichroism (CD) studies establish the stability of the overall fold, even at temperatures as high as 95ºC. A surprising loss of enzyme activity upon prolonged incubation at high temperature was observed. ESI-MS studies establish that oxidation of thiol groups of the protein may be responsible for the thermal inactivation. Chapter 5 describes the molecular structure of MjTIM, determined in collaboration with Prof. MRN Murthy’s group at the Indian Institute of Science (Gayathri et al., 2007). The crystal structure of the recombinant triosephosphate isomerase (TIM) from the archaeabacteria Methanocaldococcus jannaschii has been determined at a resolution of 2.3 Å. MjTIM is tetrameric, as suggested by solution studies and from the crystal structure, as in the case of two other structurally characterised archaeal TIMs. The archaeabacterial TIMs are shorter compared to the dimeric TIMs, with the insertions in the dimeric TIMs occurring in the vicinity of the putative tetramer interface, resulting in a hindrance to tetramerization in the dimeric TIMs. The charge distribution on the surface of archaeal TIMs also facilitates tetramerization. Analysis of the barrel interactions in TIMs suggests that these interactions are unlikely to account for the thermal stability of archaeal TIMs. A feature of the unliganded structure of MjTIM is the complete absence of electron density for the loop 6 residues. The disorder of the loop may be ascribed to a missing salt bridge between residues at the N- and C- terminal ends of the loop in MjTIM. Chapter 6 is a follow up of an interesting observation made by Vogel and Chmielewski (1994), who noticed that subtilisin cleaved rabbit muscle triosephosphate isomerase religated spontaneously upon addition of organic solvents. Further extension of this nicking and religation process with PfTIM emphasizes the importance of tertiary interactions in contributing to the stability of the (β/α)8 barrel folds (Ray et al., 1999). This chapter establishes that subtilisin nicking and religation is also facile in thermophilic MjTIM. Fragments generated by subtilisin nicking were identified using MALDI mass spectrometry at early and late stages of the cleavage for both the dimeric PfTIM and tetrameric MjTIM. This chapter also describes the comparative thermal and denaturant stability of both the enzymes. The accessibility of the Cys residues of MjTIM has been probed by examining the rates of labeling of thiol groups by iodoacetamide. The differential labeling of Cys residues has been demonstrated by mass spectrometry. Chapter 7 summarizes the main results and conclusions of the studies described in this thesis.