Abiotic stresses are responsible for limiting crop production worldwide. Among diverse abiotic stresses, drought and salinity are the most challenging. Plants under these conditions have diverse strategies for tolerating stress. Osmotic adjustment and osmoprotection occur in plants during salinity and drought stress through accumulation of compatible solutes to a high level without interfering with cellular metabolism. Polyols (sugar alcohols) including sorbitol and ribitol are one such class of compatible solutes. Using plants of wild-type (WT) and three genetically-modified lines of tomato (Solanum lycopersicum cv. ‘Ailsa Craig’), an empty vector line ‘TR22’, and 2 sdh anti-sense lines ‘TR45’, and ‘TR49’ designed to severely limit sorbitol metabolism, the objective of this work was to characterize the sorbitol cycle in tomato in response to abiotic stresses. Sorbitol and ribitol content, as well as the enzymatic activities, protein accumulation, and gene expression patterns of the key sorbitol cycle enzymes ALDOSE-6-PHOSPHATE REDUCTASE (A6PR), ALDOSE REDUCTASE (AR), and SORBITOL DEHYDROGENASE (SDH), were measured in mature leaves in response to drought stress by withholding water and by using polyethylene glycol as a root incubation solution to mimic drought stress, to salt stress by incubating roots in NaCl solution, and to incubation of roots in 100 mM sorbitol and ribitol. A6PR, not previously reported for tomato, and AR both exhibited increased activity correlated to sorbitol accumulation during the drought osmotic, and salt stresses, with SDH also increasing in WT and TR22 to metabolize sorbitol. The level of sorbitol accumulation was considerably lower than that of the common sugars glucose and fructose so was not enough to have a significant impact on tissue osmotic potential but could provide other important osmoprotective effects. Use of the sdh antisense lines indicated that SDH has the key role in sorbitol metabolism in tomato as well as a likely role in ribitol metabolism. Like sorbitol, ribitol also accumulated significantly more in the antisense lines during the stresses. Expression and/or activity of A6PR, AR, and SDH were also induced by the polyols, although it is not clear if the induction was due to a polyol signal, the osmotic effect of the incubation solution, or both. In addition, a unique post-abiotic stress phenotype was observed in the sdh anti-sense lines. After both drought and salt stresses and during a recovery phase after re-watering, the antisense lines failed to recover. This may have been due to their accumulation of ribitol. The sdh anti-sense lines were uniquely sensitive to ribitol but not sorbitol, with an apparent foliar and seed germination toxicity to ribitol. The determination that sorbitol, and perhaps ribitol as well, plays a role in abiotic responses in tomato provides a cornerstone for future studies examining how they impact tomato tolerance to abiotic stresses, and if their alteration could improve stress tolerance.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska instututet, 2006. / Härtill 4 uppsatser.

Mixed-function oxygenase systém (MFO systém) plays a vital role in the metabolism of a variety of both endogenous substrates and xenobiotics. This membrane systém consists of cytochrome P450s, NADPH:cytochrome P450 oxidoreductase (POR), cytochrome b5 and NADH:cytochrome b5 oxidoreductase (b5R). Cytochrome P450 catalyzes a monooxygenation of a substrate, while POR and cytochrome b5 represent its redox partners. Cytochrome b5, itself having a redox partner in b5R, effects the reactions catalyzed by the MFO system in various ways, through mechanisms that are not fully understood. This paper focuses on the purification of b5R and POR from rabbit liver. The microsomal fraction obtained by differential centrifugation contained 42 mg of protein per ml. From a portion of the microsomal fraction, b5R was obtained using chromatography on DEAE-Sepharose, CM-Sepharose and 5'-ADP agarose columns. The yield was 0,3 % of ferricynide-reductase activity and the product contained several contaminants in the molecular weight range of 50-70 kDa. A second purification of b5R from the microsomal fraction was carried out using a column of DEAE-Sepharose directly connected to a 5'-ADP agarose column. The b5R product was purified with a yield of 10,9 % and it once again contained several contaminants in the molecular...

Abstract Fatty acids are one of the most essential categories of biological lipids and their synthesis and degradation are vital for all organisms. Severely compromised phenotypes of yeast mutants and human patients, which have defective components in their degradative or synthetic processes for fatty acid metabolism, have highlighted the importance of these processes for overall metabolism. Most fatty acids are degraded by β-oxidation, which occurs in mitochondria and peroxisomes in mammals, whereas synthesis is catalyzed by cytosolic multifunctional peptides, although a synthesis system involving individual enzymes in mitochondria has been also proposed. In this study a novel mitochondrial 2-enoyl thioester reductase Etr1p from the yeast Candida tropicalis, its homolog Mrf1p from Saccharomyces cerevisiae, and their mammalian ortholog were identified and characterized. Observations indicating that mitochondrial localization as well as enzymatic activity is needed to complement the respiratory-deficient phenotype of the mrf1Δ strain from S. cerevisiae suggests that Etr1p and Mrf1p might act as a part of the mitochondrial fatty acid synthesis machinery, the proper function of which is essential for respiration and the maintenance of mitochondrial morphology in yeast. The mammalian enzyme, denoted Nrbf-1p, showed similar localization, enzymatic activity, and ability to rescue the growth of the mrf1Δ strain suggesting that mammals are also likely to possess the ability and required machinery for mitochondrial fatty acid synthesis. This study further included the characterization of another mitochondrial thioester reductase, 2,4-dienoyl-CoA reductase, which acts as an auxiliary enzyme in the β-oxidation of unsaturated fatty acids. The function of this gene was analyzed by creating a knock-out mouse model. While unstressed mice deficient in 2,4-dienoyl-CoA reductase were asymptomatic, metabolically challenged mice showed symptoms including hypoglycemia, hepatic steatosis, accumulation of acylcarnitines, and severe intolerance to acute cold exposure. Although the oxidation of saturated fatty acids proceeds normally, the phenotype was in many ways similar to mouse models of the disrupted classical β-oxidation pathway, except that an altered ketogenic response was not observed. This mouse model shows that a proper oxidative metabolism for unsaturated fatty acids is important for balanced fatty acid and energy metabolism.

No description available.

No description available.

Antimikrobielle Resistenzen stellen eine weltweite Herausforderung dar und sind mit einer hohen Morbidität und Mortalität verbunden. Die Letalitätsrate durch multiresistente Keime steigt stetig an, weshalb die WHO im Jahr 2017 eine Prioritätenliste resistenter Keime erstellte, die die Entwicklung neuer Antibiotika vorantreiben soll. Diese umfasst vornehmlich gramnegative Bakterien, da diese aufgrund ihres Zellaufbaus sowie diverser Resistenzmechanismen besonders widerstandsfähig gegenüber dem Angriff vieler Antibiotika sind. Einige grampositive Keime (z.B. S. aureus) stehen ebenfalls auf dieser Liste und stellen eine große Herausforderung für die Medizin dar. Infolgedessen ist die Entwicklung neuer Antiinfektiva mit neuen Angriffspunkten gegen resistente Pathogene zwingend nötig, um mit bisherigen Resistenzen umgehen zu können. Die vorliegende Arbeit beschäftigt sich mit der Entwicklung und Synthese von kovalent (reversibel) bindenden Inhibitoren der Enoyl-ACP-Reduktase FabI (Staphylococcus aureus, Escherichia coli) und der Thiolase FadA5 (Mycobacterium tuberculosis). Beide Enzyme sind essenziell für das Überleben des jeweiligen Bakteriums. FabI ist ein wichtiges und geschwindigkeitsbestimmendes Schlüsselenzym der Fettsäuresynthese Typ II diverser Bakterien. Hierbei werden wichtige Phospholipide hergestellt, die für den Aufbau der Zellmembran nötig sind. Schiebel et al. ist es gelungen, einen potenten Inhibitor für den Erreger S. aureus sowie E. coli zu entwickeln und zu charakterisieren. Ausgehend von dieser Verbindung wurde eine Substanzbibliothek mit verschiedenen „warheads“ hergestellt. Hierbei wurde die Verknüpfung zwischen dem Pyridon-Grundgerüst und der elektrophilen Gruppe sowie die über den Ether verknüpften aromatischen Ringsysteme variiert. Diese Verbindungen wurden hinsichtlich ihrer inhibitorischen Aktivität am jeweiligen Enzym getestet. Anschließend wurde von Verbindung 32 und 33, die jeweils eine gute Inhibition des Enzyms aufweisen, der IC50-Wert gemessen. Beide Verbindungen weisen eine 50-prozentige Reduktion der Enzymaktivität im mittleren nanomolaren Bereich auf. Zusätzlich wurde Verbindung 32 in einem sogenannten „jump-dilution“-Assay auf kovalente Inhibition getestet. Durch dieses Experiment konnte eine kovalente Inhibition des Enzyms ausgeschlossen werden. Die Reaktivität der eingesetzten „warheads“ wurde gegenüber einem Tripeptid mittels eines LC/MS-Iontrap-Systems bestimmt. Die untersuchten Verbindungen zeigten keine signifikante Reaktion mit der im Tripeptid eingebauten nukleophilen Aminosäure Tyrosin, deren Nukleophilie bei dem pH-Wert des Tests (pH = 8.2 und 10.8) nicht hoch genug ist. Um einen Einblick in den Bindemodus der Verbindungen zu erhalten, wurden ferner Kristallisationsversuche durchgeführt. Die erhaltenen Kristallstrukturen zeigen, dass die Verbindungen mit dem gewünschten Bindemodus am Zielenzym binden, aber eine kovalente Modifizierung des Tyrosins146 durch die eingesetzten „warheads“ aufgrund der großen Entfernung (6 Å zwischen elektrophiler Gruppe und Tyrosin146), unwahrscheinlich ist. Zusätzlich wurden die physikochemischen Eigenschaften (Stabilität, Wasserlöslichkeit und logP) der Verbindung 32 sowie Verbindung 33 charakterisiert. M. tuberculosis ist der Erreger der global verbreiteten Infektionskrankheit Tuberkulose (TB), die zu den zehn häufigsten Todesursachen weltweit gehört. Das Bakterium kann das im menschlichen Körper vorkommende Cholesterol metabolisieren und nutzt dessen Abbauprodukte als wichtige Kohlenstoffquelle. Die Thiolase FadA5 ist bei diesem Abbau ein wichtiges Enzym und konnte als potenzielles innovatives Target für neue Antibiotika definiert werden. Durch Dockingstudien konnten zwei potenzielle Leitstrukturen als Inhibitoren der Thiolase FadA5 identifiziert werden. Im Rahmen dieser Arbeit wurden die vorgeschlagenen Strukturen mit dem gewünschten „warhead“ synthetisiert und hinsichtlich ihrer inhibitorischen Aktivität gegenüber dem Enzym untersucht. Die Zielverbindungen zeigen keine signifikante Hemmung sowie kovalente Bindung über die eingesetzten „warheads“ an die Thiolase FadA5. / Antimicrobial resistance poses a global challenge and is associated with high morbidity and mortality. The case fatality rate of infections caused by multidrug-resistant pathogens continues to be on the rise, causing the WHO to compile a priority pathogens list that is supposed to advance the development of new antimicrobial compounds. The list is mainly comprised of gramnegative bacteria, since these are especially resilient to many antibiotics. This is due to their cellular structure and various mechanisms of resistance. Some grampositive bacteria are also a danger to public health and are therefore part of this list. Consequently, there is an urgent need for the development of new antiinfectives with novel modes of action, so that the current resistance situation can be adequately addressed. This work is concerned with the development and synthesis of covalent reversible inhibitors of the enoyl-ACP reductase FabI (Staphylococcus aureus, Escherichia Coli) and the thiolase FadA5 (Mycobacterium tuberculosis). Both enzymes are critically important for the survival of the respective bacteria. FabI is an essential and rate determining enzyme of the type II fatty acid synthesis of various bacteria. A number of important phospholipids required for the cell membrane are biosynthesized via this metabolic pathway. Schiebel et al. were able to develop and characterize a potent inhibitor for S. aureus and E. Coli. Using this compound as a starting point, a library of compounds carrying various “warheads” was synthesized. Further structural variations were introduced by using different linkers between the pyridone scaffold and the electrophilic group as well as diverse aromatic rings connected via the ether bridge. These compounds were assayed concerning their inhibitory activity at the respective enzyme. Of these, substances 32 and 33 showed good inhibition of the enzyme, prompting the determination of the IC50 values. The two substances were able to reduce enzymatic activity by 50% at nanomolar concentration levels. In addition, substance 32 was characterized concerning its ability to covalently inhibit its molecular target by means of the so-called jump dilution assay. This experiment showed no covalent inhibition of the target enzyme. The individual reactivity of the warhead moieties present in the library was determined against a synthetic tripeptide by using a LC/MS iontrap system. All the examined compounds showed no reaction with the nucleophilic amino acid tyrosine contained in the tripeptide at significant levels, which indicates that its nucleophilicity is insufficient at the pH of the assay (pH = 8,2 and 10,8, respectively). Crystallization experiments were conducted to ascertain the binding mode of the compounds. The crystal structures showed the substances binding to the enzyme in the desired pose, yet a covalent modification of tyrosine146 remains unlikely due to the large distance (6 Å) between the electrophilic moiety and the amino acid. Additionally, some physicochemical properties (Stability, aqueous solubility and logP) of compounds 32 and 33 were characterized. M. tuberculosis is the causative pathogen of the globally occurring infectious disease tuberculosis, which belongs to the 10 most frequently occurring causes of death worldwide. The germ is able to metabolize the cholesterol present in the human body and uses its degradation products as an important carbon source. The thiolase FadA5 is involved in this metabolic pathway and was identified as a potentially innovative target for novel antibiotics. Docking studies enabled the identification of two potential lead structures for inhibitors of FadA5. In this work, the proposed structures carrying the desired warheads were synthesized and characterized concerning their inhibitory activity at the target enzyme. The target compounds showed no significant inhibition or covalent binding to FadA5.

No description available.

Thioredoxin (Trx) and thioredoxin reductase (TrxR) along with an electron donor form a thioredoxin system. Such systems are widely distributed among the organisms belonging to the three domains of life. It is one of the major disulfide reducing systems, which provides electrons to several enzymes, such as ribonucleotide reductase, methionine sulfoxide reductase and glutathione peroxidase to name a few. It also plays an important role in combating oxidative stress and redox regulation of metabolism. Trx is a small redox protein, about 12 kDa in size, with an active site motif of Cys-X-X-Cys. The reduction of the disulfide in Trx is catalyzed by TrxR. Two types of thioredoxin reductases are known, namely NADPH thioredoxin reductase (NTR) with NADPH as the electron donor and ferredoxin thioredxoin reductase (FTR) which depends on reduced ferredoxin as electron donor. Although NTR is widely distributed in the three domains of life, it is absent in some archaea, whereas FTRs are mostly found in plants, photosynthetic eukaryotes, cyanobacteria, and some archaea. The thioredoxin system has been well studied in plants, mammals, and a few bacteria, but not much is known about the archaeal thioredoxin system. Our laboratory has been studying the thioredoxin systems of methanogenic archaea, and a major focus has been on Methanocaldococcus jannaschii, a deeply rooted archaeon that has two Trxs and one TrxR. My thesis research concerns the thioredoxin system of the late evolving members of the group which are exposed to oxygen more frequently than the deeply rooted members of the group, and have several Trxs and TrxRs. Methanosarcina mazei is one such organism, whose thioredoxin system is composed of one NTR, two FTRs, and five Trx homologs. Characterization of the components of a thioredoxin system sets the basis to further explore its function. I have expressed in Escherichia coli and purified the five Trxs and three TrxRs of M. mazei. I have shown the disulfide reductase activities in MM_Trx1 and MM_Trx5 by their ability to reduce insulin with DTT as the electron donor, and that in MM_Trx3 through the reduction of DTNB by this protein with NADPH as the electron donor, and in the presence of NTR as the enzyme. MM_Trx3 was found to be the only M. mazei thioredoxin to accept electrons through the NTR, and to form a complete Trx - NTR system. The Trx - FTR systems are well studied in plants, and such a system is yet to be defined in archaea. I have proposed a mechanism of action for one of the FTRs. FTR2 harbors a rubredoxin domain, and this unit is the only rubredoxin in this organism. Superoxide reductase, an enzyme that reduces superoxide radical to hydrogen peroxide without forming oxygen, utilizes rubredoxin as the direct electron source and this enzyme is found in certain anaerobes, including Methanosarcina species. Thus, it is possible that FTR2 provides electrons via a Trx to the superoxide reductase of M. mazei. This activity will define FTR2 as a tool in combating oxidative stress in M. mazei. In my thesis research I have laid a foundation to understand a complex thioredoxin system of M. mazei, to find the role of each Trx and TrxR, and to explore their involvement in oxidative stress and redox regulation. / Master of Science