Elevated levels of erythrocyte-conjugated dienes indicate increased lipid peroxidation in schistosomiasis mansoni patients

Schistosoma mansoni causes liver disease by inducing granulomatous inflammation. This favors formation of reactive oxygen species, including superoxide ions, hydrogen peroxide and hydroxyl radicals all of which may induce lipid peroxidation. We have evaluated lipid peroxidation in 18 patients with hepatosplenic schistosomiasis mansoni previously treated with oxamniquine followed by splenectomy, ligature of the left gastric vein and auto-implantation of spleen tissue, by measuring levels of erythrocyte-conjugated dienes and plasma malondialdehyde (MDA). Age-matched, healthy individuals (N = 18) formed the control group. Erythrocyte-conjugated dienes were extracted with dichloromethane/methanol and quantified by UV spectrophotometry, while plasma MDA was measured by reaction with thiobarbituric acid. Patient erythrocytes contained two times more conjugated dienes than control cells (584.5 ± 67.8 vs 271.7 ± 20.1 µmol/l, P < 0.001), whereas the increase in plasma MDA concentration (about 10%) was not statistically significant. These elevated conjugated dienes in patients infected by S. mansoni suggest increased lipid peroxidation in cell membranes, although this was not evident when a common marker of oxidative stress, plasma MDA, was measured. Nevertheless, these two markers of lipid peroxidation, circulating MDA and erythrocyte-conjugated dienes, correlated significantly in both patient (r = 0.62; P < 0.01) and control (r = 0.57; P < 0.05) groups. Our data show that patients with schistosomiasis have abnormal lipid peroxidation, with elevated erythrocyte-conjugated dienes implying dysfunctional cell membranes, and also imply that this may be attenuated by the redox capacity of antioxidant agents, which prevent accumulation of plasma MDA.

Reactive oxygen species and angiotensin II signaling in vascular cells: implications in cardiovascular disease

Diseases such as hypertension, atherosclerosis, hyperlipidemia, and diabetes are associated with vascular functional and structural changes including endothelial dysfunction, altered contractility and vascular remodeling. Cellular events underlying these processes involve changes in vascular smooth muscle cell (VSMC) growth, apoptosis/anoikis, cell migration, inflammation, and fibrosis. Many factors influence cellular changes, of which angiotensin II (Ang II) appears to be amongst the most important. The physiological and pathophysiological actions of Ang II are mediated primarily via the Ang II type 1 receptor. Growing evidence indicates that Ang II induces its pleiotropic vascular effects through NADPH-driven generation of reactive oxygen species (ROS). ROS function as important intracellular and intercellular second messengers to modulate many downstream signaling molecules, such as protein tyrosine phosphatases, protein tyrosine kinases, transcription factors, mitogen-activated protein kinases, and ion channels. Induction of these signaling cascades leads to VSMC growth and migration, regulation of endothelial function, expression of pro-inflammatory mediators, and modification of extracellular matrix. In addition, ROS increase intracellular free Ca2+ concentration ([Ca2+]i), a major determinant of vascular reactivity. ROS influence signaling molecules by altering the intracellular redox state and by oxidative modification of proteins. In physiological conditions, these events play an important role in maintaining vascular function and integrity. Under pathological conditions ROS contribute to vascular dysfunction and remodeling through oxidative damage. The present review focuses on the biology of ROS in Ang II signaling in vascular cells and discusses how oxidative stress contributes to vascular damage in cardiovascular disease.

Mitochondrial K+ transport and cardiac protection during ischemia/reperfusion

Mitochondrial ion transport, oxidative phosphorylation, redox balance, and physical integrity are key factors in tissue survival following potentially damaging conditions such as ischemia/reperfusion. Recent research has demonstrated that pharmacologically activated inner mitochondrial membrane ATP-sensitive K+ channels (mitoK ATP) are strongly cardioprotective under these conditions. Furthermore, mitoK ATP are physiologically activated during ischemic preconditioning, a procedure which protects against ischemic damage. In this review, we discuss mechanisms by which mitoK ATP may be activated during preconditioning and the mitochondrial and cellular consequences of this activation, focusing on end-effects which may promote ischemic protection. These effects include decreased loss of tissue ATP through reverse activity of ATP synthase due to increased mitochondrial matrix volumes and lower transport of adenine nucleotides into the matrix. MitoK ATP also decreases the release of mitochondrial reactive oxygen species by promoting mild uncoupling in concert with K+/H+ exchange. Finally, mitoK ATP activity may inhibit mitochondrial Ca2+ uptake during ischemia, which, together with decreased reactive oxygen release, can prevent mitochondrial permeability transition, loss of organelle function, and loss of physical integrity. We discuss how mitochondrial redox status, K+ transport, Ca2+ transport, and permeability transitions are interrelated during ischemia/reperfusion and are determinant factors regarding the extent of tissue damage.

Indole ring oxidation by activated leukocytes prevents the production of hypochlorous acid

Hypochlorous acid (HOCl) released by activated leukocytes has been implicated in the tissue damage that characterizes chronic inflammatory diseases. In this investigation, 14 indole derivatives, including metabolites such as melatonin, tryptophan and indole-3-acetic acid, were screened for their ability to inhibit the generation of this endogenous oxidant by stimulated leukocytes. The release of HOCl was measured by the production of taurine-chloramine when the leukocytes (2 x 10(6) cells/mL) were incubated at 37ºC in 10 mM phosphate-buffered saline, pH 7.4, for 30 min with 5 mM taurine and stimulated with 100 nM phorbol-12-myristate acetate. Irrespective of the group substituted in the indole ring, all the compounds tested including indole, 2-methylindole, 3-methylindole, 2,3-dimethylindole, 2,5-dimethylindole, 2-phenylindole, 5-methoxyindole, 6-methoxyindole, 5-methoxy-2-methylindole, melatonin, tryptophan, indole-3-acetic acid, 5-methoxy-2-methyl-3-indole-acetic acid, and indomethacin (10 µM) inhibited the chlorinating activity of myeloperoxidase (MPO) in the 23-72% range. The compounds 3-methylindole and indole-3-acetic acid were chosen as representative of indole derivatives in a dose-response study using purified MPO. The IC50 obtained were 0.10 ± 0.03 and 5.0 ± 1.0 µM (N = 13), respectively. These compounds did not affect the peroxidation activity of MPO or the production of superoxide anion by stimulated leukocytes. By following the spectral change of MPO during the enzyme turnover, the inhibition of HOCl production can be explained on the basis of the accumulation of the redox form compound-II (MPO-II), which is an inactive chlorinating species. These results show that indole derivatives are effective and selective inhibitors of MPO-chlorinating activity.

Secretion of Streptomyces tendae antifungal protein 1 by Lactococcus lactis

Lactococcus lactis, the model lactic acid bacterium, is a good candidate for heterologous protein production in both foodstuffs and the digestive tract. We attempted to produce Streptomyces tendae antifungal protein 1 (Afp1) in L. lactis with the objective of constructing a strain able to limit fungal growth. Since Afp1 activity requires disulfide bond (DSB) formation and since intracellular redox conditions are reportedly unfavorable for DSB formation in prokaryotes, Afp1 was produced as a secreted form. An inducible expression-secretion system was used to drive Afp1 secretion by L. lactis; Afp1 was fused or not with LEISSTCDA, a synthetic propeptide (LEISS) that has been described to be a secretion enhancer. Production of Afp1 alone was not achieved, but production of LEISS-Afp1 was confirmed by Western blot and immunodetection with anti-Afp1 antibodies. This protein (molecular mass: 9.8 kDa) is the smallest non-bacteriocin heterologous protein ever reported to be secreted in L. lactis via the Sec-dependent pathway. However, no anti-fungal activity was detected, even in concentrated samples of induced supernatant. This could be due to a too low secretion yield of Afp1 in L. lactis, to the absence of DSB formation, or to an improper DSB formation involving the additional cysteine residue included in LEISS propeptide. This raises questions about size limits, conformation problems, and protein secretion yields in L. lactis.

Solid-state reference and ion-selective electrodes : towards portable potentiometric sensing

Potentiometric sensors are very attractive tools for chemical analysis because of their simplicity, low power consumption and low cost. They are extensively used in clinical diagnostics and in environmental monitoring. Modern applications of both fields require improvements in the conventional construction and in the performance of the potentiometric sensors, as the trends are towards portable, on-site diagnostics and autonomous sensing in remote locations. The aim of this PhD work was to improve some of the sensor properties that currently hamper the implementation of the potentiometric sensors in modern applications. The first part of the work was concentrated on the development of a solid-state reference electrode (RE) compatible with already existing solid-contact ion-selective electrodes (ISE), both of which are needed for all-solid-state potentiometric sensing systems. A poly(vinyl chloride) membrane doped with a moderately lipophilic salt, tetrabutylammonium-tetrabutylborate (TBA-TBB), was found to show a satisfactory stability of potential in sample solutions with different concentrations. Its response time was nevertheless slow, as it required several minutes to reach the equilibrium. The TBA-TBB membrane RE worked well together with solid-state ISEs in several different situations and on different substrates enabling a miniature design. Solid contacts (SC) that mediate the ion-to-electron transduction are crucial components of well-functioning potentiometric sensors. This transduction process converting the ionic conduction of an ion-selective membrane to the electronic conduction in the circuit was studied with the help of electrochemical impedance spectroscopy (EIS). The solid contacts studied were (i) the conducting polymer (CP) poly(3,4-ethylienedioxythiophene) (PEDOT) and (ii) a carbon cloth having a high surface area. The PEDOT films were doped with a large immobile anion poly(styrene sulfonate) (PSS-) or with a small mobile anion Cl-. As could be expected, the studied PEDOT solid-contact mediated the ion-toelectron transduction more efficiently than the bare glassy carbon substrate, onto which they were electropolymerized, while the impedance of the PEDOT films depended on the mobility of the doping ion and on the ions in the electrolyte. The carbon cloth was found to be an even more effective ion-to-electron transducer than the PEDOT films and it also proved to work as a combined electrical conductor and solid contact when covered with an ion-selective membrane or with a TBA-TBB-based reference membrane. The last part of the work was focused on improving the reproducibility and the potential stability of the SC-ISEs, a problem that culminates to the stability of the standard potential E°. It was proven that the E° of a SC-ISE with a conducting polymer as a solid contact could be adjusted by reducing or oxidizing the CP solid contact by applying current pulses or a potential to it, as the redox state of the CP solid-contact influences the overall potential of the ISE. The slope and thus the analytical performance of the SC-ISEs were retained despite the adjustment of the E°. The shortcircuiting of the SC-ISE with a conventional large-capacitance RE was found to be a feasible instrument-free method to control the E°. With this method, the driving force for the oxidation/reduction of the CP was the potential difference between the RE and the SC-ISE, and the position of the adjusted potential could be controlled by choosing a suitable concentration for the short-circuiting electrolyte. The piece-to-piece reproducibility of the adjusted potential was promising, and the day-today reproducibility for a specific sensor was excellent. The instrumentfree approach to control the E° is very attractive considering practical applications.

TRP channels, omega-3 fatty acids, and oxidative stress in neurodegeneration: from the cell membrane to intracellular cross-links

The transient receptor potential channels family (TRP channels) is a relatively new group of cation channels that modulate a large range of physiological mechanisms. In the nervous system, the functions of TRP channels have been associated with thermosensation, pain transduction, neurotransmitter release, and redox signaling, among others. However, they have also been extensively correlated with the pathogenesis of several innate and acquired diseases. On the other hand, the omega-3 polyunsaturated fatty acids (n-3 fatty acids) have also been associated with several processes that seem to counterbalance or to contribute to the function of several TRPs. In this short review, we discuss some of the remarkable new findings in this field. We also review the possible roles played by n-3 fatty acids in cell signaling that can both control or be controlled by TRP channels in neurodegenerative processes, as well as both the direct and indirect actions of n-3 fatty acids on TRP channels.

2-Bromo-1,4-naphthoquinone: a potentially improved substitute of menadione in Apatone™ therapy

Apatone™, a combination of menadione (2-methyl-1,4-naphthoquinone, VK3) and ascorbic acid (vitamin C, VC) is a new strategy for cancer treatment. Part of its effect on tumor cells is related to the cellular pro-oxidative imbalance provoked by the generation of hydrogen peroxide (H2O2) through naphthoquinone redox cycling. In this study, we attempted to find new naphthoquinone derivatives that would increase the efficiency of H2O2 production, thereby potentially increasing its efficacy for cancer treatment. The presence of an electron-withdrawing group in the naphthoquinone moiety had a direct effect on the efficiency of H2O2 production. The compound 2-bromo-1,4-naphthoquinone (BrQ), in which the bromine atom substituted the methyl group in VK3, was approximately 10- and 19-fold more efficient than VK3 in terms of oxygen consumption and H2O2 production, respectively. The ratio [H2O2]produced / [naphthoquinone]consumed was 68 ± 11 and 5.8 ± 0.2 (µM/µM) for BrQ and VK3, respectively, indicating a higher efficacy of BrQ as a catalyst for the autoxidation of ascorbic acid. Both VK3 and BrQ reacted with glutathione (GSH), but BrQ was the more effective substrate. Part of GSH was incorporated into the naphthoquinone, producing a nucleophilic substitution product (Q-SG). The depletion of BrQ by GSH did not prevent its redox capacity since Q-SG was also able to catalyze the production of reactive oxygen species. VK3/VC has already been submitted to clinical trials for the treatment of prostate cancer and has demonstrated promising results. However, replacement of VK3 with BrQ will open new lines of investigation regarding this approach to cancer treatment.

Target-directed catalytic metallodrugs

Most drugs function by binding reversibly to specific biological targets, and therapeutic effects generally require saturation of these targets. One means of decreasing required drug concentrations is incorporation of reactive metal centers that elicit irreversible modification of targets. A common approach has been the design of artificial proteases/nucleases containing metal centers capable of hydrolyzing targeted proteins or nucleic acids. However, these hydrolytic catalysts typically provide relatively low rate constants for target inactivation. Recently, various catalysts were synthesized that use oxidative mechanisms to selectively cleave/inactivate therapeutic targets, including HIV RRE RNA or angiotensin converting enzyme (ACE). These oxidative mechanisms, which typically involve reactive oxygen species (ROS), provide access to comparatively high rate constants for target inactivation. Target-binding affinity, co-reactant selectivity, reduction potential, coordination unsaturation, ROS products (metal-associated vsmetal-dissociated; hydroxyl vs superoxide), and multiple-turnover redox chemistry were studied for each catalyst, and these parameters were related to the efficiency, selectivity, and mechanism(s) of inactivation/cleavage of the corresponding target for each catalyst. Important factors for future oxidative catalyst development are 1) positioning of catalyst reduction potential and redox reactivity to match the physiological environment of use, 2) maintenance of catalyst stability by use of chelates with either high denticity or other means of stabilization, such as the square planar geometric stabilization of Ni- and Cu-ATCUN complexes, 3) optimal rate of inactivation of targets relative to the rate of generation of diffusible ROS, 4) targeting and linker domains that afford better control of catalyst orientation, and 5) general bio-availability and drug delivery requirements.

Involvement of Src tyrosine kinase and protein kinase C in the expression of macrophage migration inhibitory factor induced by H2O2 in HL-1 mouse cardiac muscle cells

Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, plays an important role in the pathogenesis of atrial fibrillation; however, the upstream regulation of MIF in atrial myocytes remains unclear. In the present study, we investigated whether and how MIF is regulated in response to the renin-angiotensin system and oxidative stress in atrium myocytes (HL-1 cells). MIF protein and mRNA levels in HL-1 cells were assayed using immunofluorescence, real-time PCR, and Western blot. The result indicated that MIF was expressed in the cytoplasm of HL-1 cells. Hydrogen peroxide (H2O2), but not angiotensin II, stimulated MIF expression in HL-1 cells. H2O2-induced MIF protein and gene levels increased in a dose-dependent manner and were completely abolished in the presence of catalase. H2O2-induced MIF production was completely inhibited by tyrosine kinase inhibitors genistein and PP1, as well as by protein kinase C (PKC) inhibitor GF109203X, suggesting that redox-sensitive MIF production is mediated through tyrosine kinase and PKC-dependent mechanisms in HL-1 cells. These results suggest that MIF is upregulated by HL-1 cells in response to redox stress, probably by the activation of Src and PKC.

Therapeutic concentration of morphine reduces oxidative stress in glioma cell line

Morphine is a potent analgesic opioid used extensively for pain treatment. During the last decade, global consumption grew more than 4-fold. However, molecular mechanisms elicited by morphine are not totally understood. Thus, a growing literature indicates that there are additional actions to the analgesic effect. Previous studies about morphine and oxidative stress are controversial and used concentrations outside the range of clinical practice. Therefore, in this study, we hypothesized that a therapeutic concentration of morphine (1 μM) would show a protective effect in a traditional model of oxidative stress. We exposed the C6 glioma cell line to hydrogen peroxide (H2O2) and/or morphine for 24 h and evaluated cell viability, lipid peroxidation, and levels of sulfhydryl groups (an indicator of the redox state of the cell). Morphine did not prevent the decrease in cell viability provoked by H2O2 but partially prevented lipid peroxidation caused by 0.0025% H2O2 (a concentration allowing more than 90% cell viability). Interestingly, this opioid did not alter the increased levels of sulfhydryl groups produced by exposure to 0.0025% H2O2, opening the possibility that alternative molecular mechanisms (a direct scavenging activity or the inhibition of NAPDH oxidase) may explain the protective effect registered in the lipid peroxidation assay. Our results demonstrate, for the first time, that morphine in usual analgesic doses may contribute to minimizing oxidative stress in cells of glial origin. This study supports the importance of employing concentrations similar to those used in clinical practice for a better approximation between experimental models and the clinical setting.

Caryocar brasiliense camb protects against genomic and oxidative damage in urethane-induced lung carcinogenesis

The antioxidant effects of Caryocar brasiliense Camb, commonly known as the pequi fruit, have not been evaluated to determine their protective effects against oxidative damage in lung carcinogenesis. In the present study, we evaluated the role of pequi fruit against urethane-induced DNA damage and oxidative stress in forty 8-12 week old male BALB/C mice. An in vivo comet assay was performed to assess DNA damage in lung tissues and changes in lipid peroxidation and redox cycle antioxidants were monitored for oxidative stress. Prior supplementation with pequi oil or its extract (15 µL, 60 days) significantly reduced urethane-induced oxidative stress. A protective effect against DNA damage was associated with the modulation of lipid peroxidation and low protein and gene expression of nitric oxide synthase. These findings suggest that the intake of pequi fruit might protect against in vivo genotoxicity and oxidative stress.

Vanadiiniakut

Nykyään monet käyttökohteet vaativat akuilta aiempaa parempaa suorituskykyä, joten on syntynyt kasvavaa tarvetta uusien akkujen kehittämiselle erilaisista uusista materiaaleista ja uusiin valmistusmenetelmiin pohjautuen. Työn tavoitteena on selvittää kirjallisuustutkimuksena litium-vanadiini-fosfaattiakun ja vanadiini-redoksi-virtausakun ominaisuuksia, saatavuutta, sovelluskohteita ja vertailla niitä muutamaan eri litiumakkutekniikkaan. Tutkimuksen perusteella litium-vanadiini-fosfaattiakkuja ei ole vielä saatavilla kaupallisesti, joten työssä tutkittiin niitä teknisten raporttien pohjalta. Raporttien pohjalta arvioituna, parhaita ominaisuuksia litium-vanadiini-fosfaattiakuilla on erinomainen kuormituksen kesto ja korkea nimellisjännite. Sähköisissä ajoneuvoissa litium-vanadiini-fosfaattiakuilla on suurimmat mahdollisuudet erilaisissa hybridiajoneuvoissa, mutta todennäköisesti ne soveltuvat täyssähköisiin ajoneuvoihin vähintään yhtä hyvin kuin esimerkiksi litium-rauta-fosfaattiakut, jos valmistuskustannukset olisivat samalla tasolla. Vanadiini-redoksi-virtausakkua on jo markkinoilla usean valmistajan toimesta. Niiden ominaisuudet poikkeavat paljon muista akkutyypeistä ja erikoisuutena on mahdollisuus akun pikalataukseen elektrolyyttinesteet vaihtamalla. Syklien kestossa päästään myös erinomaisiin arvoihin, mutta suurimmat ongelmat ovat lyijyakkuakin matalampi energiatiheys ja tehotiheys. Vanadiini-redoksi-virtausakut soveltuvat parhaiten suuren kokoluokan sähköverkkoratkaisuihin ja sähköisissä ajoneuvoissa niiden mahdollisuudet rajoittunevat täyssähköisiin linja-autoihin ja isoihin työkoneisiin.

Alternative electron transfer routes involved in photoprotection of cyanobacteria

In oxygenic photosynthesis, the highly oxidizing reactions of water splitting produce reactive oxygen species (ROS) and other radicals that could damage the photosynthetic apparatus and affect cell viability. Under particular environmental conditions, more electrons are produced in water oxidation than can be harmlessly used by photochemical processes for the reduction of metabolic electron sinks. In these circumstances, the excess of electrons can be delivered, for instance, to O2, resulting in the production of ROS. To prevent detrimental reactions, a diversified assortment of photoprotection mechanisms has evolved in oxygenic photosynthetic organisms. In this thesis, I focus on the role of alternative electron transfer routes in photoprotection of the cyanobacterium Synechocystis sp. PCC 6803. Firstly, I discovered a novel subunit of the NDH-1 complex, NdhS, which is necessary for cyclic electron transfer around Photosystem I, and provides tolerance to high light intensities. Cyclic electron transfer is important in modulating the ATP/NADPH ratio under stressful environmental conditions. The NdhS subunit is conserved in many oxygenic phototrophs, such as cyanobacteria and higher plants. NdhS has been shown to link linear electron transfer to cyclic electron transfer by forming a bridge for electrons accumulating in the Ferredoxin pool to reach the NDH-1 complexes. Secondly, I thoroughly investigated the role of the entire flv4-2 operon in the photoprotection of Photosystem II under air level CO2 conditions and varying light intensities. The operon encodes three proteins: two flavodiiron proteins Flv2 and Flv4 and a small Sll0218 protein. Flv2 and Flv4 are involved in a novel electron transport pathway diverting electrons from the QB pocket of Photosystem II to electron acceptors, which still remain unknown. In my work, it is shown that the flv4-2 operon-encoded proteins safeguard Photosystem II activity by sequestering electrons and maintaining the oxidized state of the PQ pool. Further, Flv2/Flv4 was shown to boost Photosystem II activity by accelerating forward electron flow, triggered by an increased redox potential of QB. The Sll0218 protein was shown to be differentially regulated as compared to Flv2 and Flv4. Sll0218 appeared to be essential for Photosystem II accumulation and was assigned a stabilizing role for Photosystem II assembly/repair. It was also shown to be responsible for optimized light-harvesting. Thus, Sll0218 and Flv2/Flv4 cooperate to protect and enhance Photosystem II activity. Sll0218 ensures an increased number of active Photosystem II centers that efficiently capture light energy from antennae, whilst the Flv2/Flv4 heterodimer provides a higher electron sink availability, in turn, promoting a safer and enhanced activity of Photosystem II. This intertwined function was shown to result in lowered singlet oxygen production. The flv4-2 operon-encoded photoprotective mechanism disperses excess excitation pressure in a complimentary manner with the Orange Carotenoid Protein-mediated non-photochemical quenching. Bioinformatics analyses provided evidence for the loss of the flv4-2 operon in the genomes of cyanobacteria that have developed a stress inducible D1 form. However, the occurrence of various mechanisms, which dissipate excitation pressure at the acceptor side of Photosystem II was revealed in evolutionarily distant clades of organisms, i.e. cyanobacteria, algae and plants.

Monooxygenase Diversity and Oxygen Activation within Polyketide Antibiotic Biosynthesis

Molecular oxygen (O2) is a key component in cellular respiration and aerobic life. Through the redox potential of O2, the amount of free energy available to organisms that utilize it is greatly increased. Yet, due to the nature of the O2 electron configuration, it is non-reactive to most organic molecules in the ground state. For O2 to react with most organic compounds it must be activated. By activating O2, oxygenases can catalyze reactions involving oxygen incorporation into organic compounds. The oxygen activation mechanisms employed by many oxygenases to have been studied, and they often include transition metals and selected organic compounds. Despite the diversity of mechanisms for O2 activation explored in this thesis, all of the monooxygenases studied in the experimental part activate O2 through a transient carbanion intermediate. One of these enzymes is the small cofactorless monooxygenase SnoaB. Cofactorless monooxygenases are unusual oxygenases that require neither transition metals nor cofactors to activate oxygen. Based on our biochemical characterization and the crystal structure of this enzyme, the mechanism most likely employed by SnoaB relies on a carbanion intermediate to activate oxygen, which is consistent with the proposed substrate-assisted mechanism for this family of enzymes. From the studies conducted on the two-component system AlnT and AlnH, both the functions of the NADH-dependent flavin reductase, AlnH, and the reduced flavin dependent monooxygenase, AlnT, were confirmed. The unusual regiochemistry proposed for AlnT was also confirmed on the basis of the structure of a reaction product. The mechanism of AlnT, as with other flavin-dependent monooxygenases, is likely to involve a caged radical pair consisting of a superoxide anion and a neutral flavin radical formed from an initial carbanion intermediate. In the studies concerning the engineering of the S-adenosyl-L-methionine (SAM) dependent 4-O-methylase DnrK and the homologous atypical 10-hydroxylase RdmB, our data suggest that an initial decarboxylation of the substrate is catalyzed by both of these enzymes, which results in the generation of a carbanion intermediate. This intermediate is not essential for the 4-O-methylation reaction, but it is important for the 10-hydroxylation reaction, since it enables substrate-assisted activation of molecular oxygen involving a single electron transfer to O2 from a carbanion intermediate. The only role for SAM in the hydroxylation reaction is likely to be stabilization of the carbanion through the positive charge of the cofactor. Based on the DnrK variant crystal structure and the characterizations of several DnrK variants, the insertion of a single amino acid in DnrK (S297) is sufficient for gaining a hydroxylation function, which is likely caused by carbanion stabilization through active site solvent restriction. Despite large differences in the three-dimensional structures of the oxygenases and the potential for multiple oxygen activation mechanisms, all the enzymes in my studies rely on carbanion intermediates to activate oxygen from either flavins or their substrates. This thesis provides interesting examples of divergent evolution and the prevalence of carbanion intermediates within polyketide biosynthesis. This mechanism appears to be recurrent in aromatic polyketide biosynthesis and may reflect the acidic nature of these compounds, propensity towards hydrogen bonding and their ability to delocalize π-electrons.