Reduced Flavin Adenine Dinucleotide Research Articles

Theoretical insights into the formation of oxygen activation sites with the affection of protein microenvironment in Flavin-Dependent monooxygenases MsuD

Flavin-dependent monooxygenases are very famous for their capability to catalyze the oxidation of various substrates using oxygen and reduced flavin mononucleotide (FMNH-) or reduced flavin adenine dinucleotide (FADH-). When investigating the catalytic mechanism of flavin-dependent monooxygenase systems, a fundamental problem remains unsolved is that the O2 binding scheme within the reaction cavity is unclear. By applying molecular dynamics simulations, MDpocket simulations, residue mutation molecular dynamics simulation and energy decomposition analyses, this work identified the O2 binding pockets and discovered a possible oxygen migration channel within MsuD enzyme. Molecular dynamics simulations after residue mutation proved that residues Asn104 plays an important role in maintaining the O2 position within the reaction cavity. By analyzing the interaction between O2 and the surrounding protein microenvironment, we demonstrated that N5 is a more favored reaction site and speculated a reasonable O2 activation process. This work recovers the formation of oxygen activation sites in MsuD enzyme, which is of great significance to further investigations of the catalytic reaction mechanism. This work provides a feasible method to study the O2 binding in proteins and the role of the protein microenvironment around the reaction cavity.

The dynamics of the flavin, NADPH, and active site loops determine the mechanism of activation of class B flavin-dependent monooxygenases.

Flavin-dependent monooxygenases (FMOs) constitute a diverse enzyme family that catalyzes crucial hydroxylation, epoxidation, and Baeyer-Villiger reactions across various metabolic pathways in all domains of life. Due to the intricate nature of this enzyme family's mechanisms, some aspects of their functioning remain unknown. Here, we present the results of molecular dynamics computations, supplemented by a bioinformatics analysis, that clarify the early stages of their catalytic cycle. We have elucidated the intricate binding mechanism of NADPH and L-Orn to a class B monooxygenase, the ornithine hydroxylase from known as SidA. Our investigation involved a comprehensive characterization of the conformational changes associated with the FAD (Flavin Adenine Dinucleotide) cofactor, transitioning from the out to the in position. Furthermore, we explored the rotational dynamics of the nicotinamide ring of NADPH, shedding light on its role in facilitating FAD reduction, supported by experimental evidence. Finally, we also analyzed the extent of conservation of two Tyr-loops that play critical roles in the process.

Dynamic interplays between three redox cofactors in a DNA photolyase revealed by spectral decomposition

DNA repair catalyzed by photolyases is accomplished by a light-dependent electron transfer event from a fully reduced flavin adenine dinucleotide to a DNA lesion site. Prokaryotic DNA photolyase, PhrB, possesses a ribolumazine cofactor and a four-iron-four-sulfur cluster in addition to the catalytic flavin, but their functional roles are poorly understood. Here, we employ time-resolved absorption spectroscopy to probe light-induced responses in both solution and single crystals of PhrB. We jointly analyze a large collection of light-induced difference spectra from the wild-type and mutant PhrB obtained under different light and redox conditions. By applying singular value decomposition to 159 time series, we dissect light-induced spectral changes and examine the dynamic interplay between three cofactors. Our findings suggest that these cofactors form an interdependent redox network to coordinate light-induced redox responses. We propose that the ribolumazine cofactor serves as a photoprotective pigment under intense light or prolonged illumination, while the iron-sulfur cluster acts as a transient electron cache to maintain balance between two otherwise independent photoreactions of the flavin and ribolumazine.

The reductive half-reaction of two bifurcating electron-transferring flavoproteins: Evidence for changes in flavin reduction potentials mediated by specific conformational changes

The EtfAB components of two bifurcating flavoprotein systems, the crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase from the bacterium Megasphaera elsdenii and the menaquinone-dependent NADH:ferredoxin oxidoreductase from the archaeon Pyrobaculum aerophilum, have been investigated. With both proteins, we find that removal of the electron-transferring flavin adenine dinucleotide (FAD) moiety from both proteins results in an uncrossing of the reduction potentials of the remaining bifurcating FAD; this significantly stabilizes the otherwise very unstable semiquinone state, which accumulates over the course of reductive titrations with sodium dithionite. Furthermore, reduction of both EtfABs depleted of their electron-transferring FAD by NADH was monophasic with a hyperbolic dependence of reaction rate on the concentration of NADH. On the other hand, NADH reduction of the replete proteins containing the electron-transferring FAD was multiphasic, consisting of a fast phase comparable to that seen with the depleted proteins followed by an intermediate phase that involves significant accumulation of FAD⋅−, again reflecting uncrossing of the half-potentials of the bifurcating FAD. This is then followed by a slow phase that represents the slow reduction of the electron-transferring FAD to FADH−, with reduction of the now fully reoxidized bifurcating FAD by a second equivalent of NADH. We suggest that the crossing and uncrossing of the reduction half-potentials of the bifurcating FAD is due to specific conformational changes that have been structurally characterized.

DASH cryptochrome 1, a UV-A receptor, balances the photosynthetic machinery of Chlamydomonas reinhardtii.

Drosophila, Arabidopsis, Synechocystis, Homo (DASH) cryptochromes belong to the cryptochrome/photolyase family and can act as DNA repair enzymes. In bacteria and fungi, they also can play regulatory roles, but in plants their biological functions remain elusive. Here, we characterize CRY-DASH1 from the green alga Chlamydomonas reinhardtii. We perform biochemical and invitro photochemical analysis. For functional characterization, a knock-out mutant of cry-dash1 is used. CRY-DASH1 protein is localized in the chloroplast and accumulates at midday. Although the photoautotrophic growth of the mutant is significantly reduced compared to the wild-type (WT), the mutant has increased levels of photosynthetic pigments and a higher maximum photochemical efficiency of photosystem II (PS II). Hyper-stacking of thylakoid membranes occurs together with an increase in proteins of the PS II reaction center, D1 and its antenna CP43, but not of their transcripts. CRY-DASH1 binds fully reduced flavin adenine dinucleotide and the antenna 5,10-methenyltetrahydrofolate, leading to an absorption peak in the UV-A range. Supplementation of white light with UV-A increases photoautotrophic growth of the WT but not of the cry-dash1 mutant. These results suggest a balancing function of CRY-DASH1 in the photosynthetic machinery and point to its role as a photoreceptor for the UV-A range separated from the absorption of photosynthetic pigments.

Ultrafast dynamics of fully reduced flavin in catalytic structures of thymidylate synthase ThyX.

Thymidylate is a vital DNA precursor synthesized by thymidylate synthases. ThyX is a flavin-dependent thymidylate synthase found in several human pathogens and absent in humans, which makes it a potential target for antimicrobial drugs. This enzyme methylates the 2'-deoxyuridine 5'-monophosphate (dUMP) to 2'-deoxythymidine 5'-monophosphate (dTMP) using a reduced flavin adenine dinucleotide (FADH-) as prosthetic group and (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2THF) as a methylene donor. Recently, it was shown that ThyX-catalyzed reaction is a complex process wherein FADH- promotes both methylene transfer and reduction of the transferred methylene into a methyl group. Here, we studied the dynamic and photophysics of FADH- bound to ThyX, in several substrate-binding states (no substrate, in the presence of dUMP or folate or both) by femtosecond transient absorption spectroscopy. This methodology provides valuable information about the ground-state configuration of the isoalloxazine moiety of FADH- and the rigidity of its local environment, through spectra shape and excited-state lifetime parameters. In the absence of substrate, the environment of FADH- in ThyX is only mildly more constrained than that of free FADH- in solution. The addition of dUMP however narrows the distribution of ground-state configurations and increases the constraints on the butterfly bending motion in the excited state. Folate binding results in the selection of new ground-state configurations, presumably located at a greater distance from the conical intersection where excited-state decay occurs. When both substrates are present, the ground-state configuration appears on the contrary rather limited to a geometry close to the conical intersection, which explains the relatively fast excited-state decay (100 ps on the average), even if the environment of the isoalloxazine is densely packed. Hence, although the environment of the flavin is dramatically constrained, FADH- retains a dynamic necessary to shuttle carbon from folate to dUMP. Our study demonstrates the high sensitivity of FADH- photophysics to the constraints exerted by its immediate surroundings.

Intensifying Electron Utilization by Surface-Anchored Rh Complex for Enhanced Nicotinamide Cofactor Regeneration and Photoenzymatic CO2 Reduction.

Solar-driven photocatalytic regeneration of cofactors, including reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), and reduced flavin adenine dinucleotide (FADH2), could ensure the sustainable energy supply of enzymatic reactions catalyzed by oxidoreductases for the efficient synthesis of chemicals. However, the elevation of cofactor regeneration efficiency is severely hindered by the inefficient utilization of electrons transferred on the surface of photocatalysts. Inspired by the phenomenon of ferredoxin-NADP+ reductase (FNR) anchoring on thylakoid membrane, herein, a homogeneous catalyst of rhodium (Rh) complex, [Cp∗Rh(bpy)H2O]2+, was anchored on polymeric carbon nitride (PCN) mediated by a tannic acid/polyethyleneimine (TA/PEI) adhesive layer, acquiring PCN@TA/PEI-Rh core@shell photocatalyst. Illuminated by visible light, electrons were excited from the PCN core, then transferred through the TA/PEI shell, and finally captured by the surface-anchored Rh for instant utilization during the regeneration of NADH. The TA/PEI-Rh shell could facilitate the electron transfer from the PCN core and, more importantly, achieved ~1.3-fold elevation of electron utilization efficiency compared with PCN. Accordingly, the PCN@TA/PEI-Rh afforded the NADH regeneration efficiency of 37.8% after 20 min reaction under LED light (405 nm) illumination, over 1.5 times higher than PCN with free Rh. Coupling of the NADH regeneration system with formate dehydrogenase achieved continuous production of formate from carbon dioxide (CO2). Our study may provide a generic and effective strategy to elevate the catalytic efficiency of a photocatalyst through intensifying the electron utilization.

Dissecting the low catalytic capability of flavin-dependent halogenases

Although flavin-dependent halogenases (FDHs) are attractive biocatalysts, their practical applications are limited because of their low catalytic efficiency. Here, we investigated the reaction mechanisms and structures of tryptophan 6-halogenase (Thal) from Streptomyces albogriseolus using stopped-flow, rapid-quench flow, quantum/mechanics molecular mechanics calculations, crystallography, and detection of intermediate (hypohalous acid [HOX]) liberation. We found that the key flavin intermediate, C4a-hydroperoxyflavin (C4aOOH-FAD), formed by Thal and other FDHs (tryptophan 7-halogenase [PrnA] and tryptophan 5-halogenase [PyrH]), can react with I−, Br−, and Cl− but not F− to form C4a-hydroxyflavin and HOX. Our experiments revealed that I− reacts with C4aOOH-FAD the fastest with the lowest energy barrier and have shown for the first time that a significant amount of the HOX formed leaks out as free HOX. This leakage is probably a major cause of low product coupling ratios in all FDHs. Site-saturation mutagenesis of Lys79 showed that changing Lys79 to any other amino acid resulted in an inactive enzyme. However, the levels of liberated HOX of these variants are all similar, implying that Lys79 probably does not form a chloramine or bromamine intermediate as previously proposed. Computational calculations revealed that Lys79 has an abnormally lower pKa compared with other Lys residues, implying that the catalytic Lys may act as a proton donor in catalysis. Analysis of new X-ray structures of Thal also explains why premixing of FDHs with reduced flavin adenine dinucleotide generally results in abolishment of C4aOOH-FAD formation. These findings reveal the hidden factors restricting FDHs capability which should be useful for future development of FDHs applications.

Not Cleaving the His-tag of Thal Results in More Tightly Packed and Better-Diffracting Crystals

Flavin-dependent halogenases chlorinate or brominate their substrates in an environmentally friendly manner, only requiring the cofactor reduced flavin adenine dinucleotide (FADH2), oxygen, and halide salts. The tryptophan 6-halogenase Thal exhibits two flexible loops, which become ordered (substrate-binding loop) or adopt a closed conformation (FAD loop) upon substrate or cofactor binding. Here, we describe the structure of NHis-Thal-RebH5 containing an N-terminal His-tag from pET28a, which crystallized in a different space group (P21) and, surprisingly, diffracted to a higher resolution of 1.63 Å than previously deposited Thal structures (P64; ~2.2 Å) with cleaved His-tag. Interestingly, the binding of glycine in the active site can induce an ordered conformation of the substrate-binding loop.

Computational Modeling Analysis of Generation of Reactive Oxygen Species by Mitochondrial Assembled and Disintegrated Complex II.

Reactive oxygen species (ROS) function as critical mediators in a broad range of cellular signaling processes. The mitochondrial electron transport chain is one of the major contributors to ROS formation in most cells. Increasing evidence indicates that the respiratory Complex II (CII) can be the predominant ROS generator under certain conditions. A computational, mechanistic model of electron transfer and ROS formation in CII was developed in the present study to facilitate quantitative analysis of mitochondrial ROS production. The model was calibrated by fitting the computer simulated results to experimental data obtained on submitochondrial particles (SMP) prepared from bovine and rat heart mitochondria upon inhibition of the ubiquinone (Q)-binding site by atpenin A5 (AA5) and Complex III by myxothiazol, respectively. The model predicts that only reduced flavin adenine dinucleotide (FADH2) in the unoccupied dicarboxylate state and flavin semiquinone radical (FADH•) feature the experimentally observed bell-shaped dependence of the rate of ROS production on the succinate concentration upon inhibition of respiratory Complex III (CIII) or Q-binding site of CII, i.e., suppression of succinate-Q reductase (SQR) activity. The other redox centers of CII such as Fe-S clusters and Q-binding site have a hyperbolic dependence of ROS formation on the succinate concentration with very small maximal rate under any condition and cannot be considered as substantial ROS generators in CII. Computer simulation results show that CII disintegration (which results in dissociation of the hydrophilic SDHA/SDHB subunits from the inner membrane to the mitochondrial matrix) causes crucial changes in the kinetics of ROS production by CII that are qualitatively and quantitatively close to changes in the kinetics of ROS production by assembled CII upon inhibition of CIII or Q-binding site of CII. Thus, the main conclusions from the present computational modeling study are the following: (i) the impairment of the SQR activity of CII resulting from inhibition of CIII or Q-binding site of CII and (ii) CII disintegration causes a transition in the succinate-dependence of ROS production from a small-amplitude sigmoid (hyperbolic) shape, determined by Q-binding site or [3Fe-4S] cluster to a high-amplitude bell-shaped kinetics with a shift to small subsaturated concentrations of succinate, determined by the flavin site.

Mechanobiology of Cartilage Impact Via Real-Time Metabolic Imaging.

Cartilage loading is important in both structural and biological contexts, with overloading known to cause osteoarthritis (OA). Cellular metabolism, which can be evaluated through the relative measures of glycolysis and oxidative phosphorylation, is important in disease processes across tissues. Details of structural damage coupled with cellular metabolism in cartilage have not been evaluated. Therefore, the aim of this study was to characterize the time- and location-dependent metabolic response to traumatic impact loading in articular cartilage. Cartilage samples from porcine femoral condyles underwent a single traumatic injury that created cracks in most samples. Before and up to 30 min after loading, samples underwent optical metabolic imaging. Optical metabolic imaging measures the fluorescent intensity of byproducts of the two metabolic pathways, flavin adenine dinucleotide for oxidative phosphorylation and nicotinamide adenine dinucleotide ± phosphatefor glycolysis, as well as the redox ratio between them. Images were taken at varied distances from the center of the impact. Shortly after impact, fluorescence intensity in both channels decreased, while redox ratio was unchanged. The most dramatic metabolic response was measured closest to the impact center, with suppressed fluorescence in both channels relative to baseline. Redox ratio varied nonlinearly as a function of distance from the impact. Finally, both lower and higher magnitude loading reduced flavin adenine dinucleotide fluorescence, whereas reduced nicotinamide adenine dinucleotide ± phosphatefluorescence was associated only with low strain loads and high contact pressure loads, respectively. In conclusion, this study performed novel analysis of metabolic activity following induction of cartilage damage and demonstrated time-, distance-, and load-dependent response to traumatic impact loading.

Enantioselective Epoxidation by Flavoprotein Monooxygenases Supported by Organic Solvents

Styrene and indole monooxygenases (SMO and IMO) are two-component flavoprotein monooxygenases composed of a nicotinamide adenine dinucleotide (NADH)-dependent flavin adenine dinucleotide (FAD)-reductase (StyB or IndB) and a monooxygenase (StyA or IndA). The latter uses reduced FAD to activate oxygen and to oxygenate the substrate while releasing water. We circumvented the need for the reductase by direct FAD reduction in solution using the NAD(P)H-mimic 1-benzyl-1,4-dihydronicotinamide (BNAH) to fuel monooxygenases without NADH requirement. Herein, we report on the hitherto unknown solvent tolerance for the indole monooxygenase from Gemmobacter nectariphilus DSM15620 (GnIndA) and the styrene monooxygenase from Gordonia rubripertincta CWB2 (GrStyA). These enzymes were shown to convert bulky and rather hydrophobic styrene derivatives in the presence of organic cosolvents. Subsequently, BNAH-driven biotransformation was furthermore optimized with regard to the applied cosolvent and its concentration as well as FAD and BNAH concentration. We herein demonstrate that GnIndA and GrStyA enable selective epoxidations of allylic double bonds (up to 217 mU mg−1) in the presence of organic solvents such as tetrahydrofuran, acetonitrile, or several alcohols. Notably, GnIndA was found to resist methanol concentrations up to 25 vol.%. Furthermore, a diverse substrate preference was determined for both enzymes, making their distinct use very interesting. In general, our results seem representative for many IMOs as was corroborated by in silico mutagenetic studies.

Binding of FAD and tryptophan to the tryptophan 6-halogenase Thal is negatively coupled.

Flavin‐dependent halogenases require reduced flavin adenine dinucleotide (FADH2), O2, and halide salts to halogenate their substrates. We describe the crystal structures of the tryptophan 6‐halogenase Thal in complex with FAD or with both tryptophan and FAD. If tryptophan and FAD were soaked simultaneously, both ligands showed impaired binding and in some cases only the adenosine monophosphate or the adenosine moiety of FAD was resolved, suggesting that tryptophan binding increases the mobility mainly of the flavin mononucleotide moiety. This confirms a negative cooperativity between the binding of substrate and cofactor that was previously described for other tryptophan halogenases. Binding of substrate to tryptophan halogenases reduces the affinity for the oxidized cofactor FAD presumably to facilitate the regeneration of FADH2 by flavin reductases.

Structural and Functional Characterization of 4-Hydroxyphenylacetate 3-Hydroxylase from Escherichia coli.

The hydroxylation of phenols into polyphenols, which are valuable chemicals and pharmaceutical products, is a challenging reaction. The search for green synthetic processes has led to considering microorganisms and pure hydroxylases as catalysts for phenol hydroxylation. Herein, we report the structural and functional characterization of the flavin adenine dinucleotide (FAD)-dependent 4-hydroxyphenylacetate 3-monooxygenase from Escherichia coli, named HpaB. It is shown that this enzyme enjoys a relatively broad substrate specificity, which allows the conversion of a number of non-natural phenolic compounds, such as tyrosol, hydroxymandelic acid, coumaric acid, hydroxybenzoic acid and its methyl ester, and phenol, into the corresponding catechols. The reaction can be performed by using a simple chemical assay based on formate as the electron donor and the organometallic complex [Rh(bpy)Cp*(H2 O)]2+ (Cp*: 1,2,3,4,5-pentamethylcyclopentadiene, bpy: 2,2'-bipyridyl) as the catalyst for FAD reduction. The availability of a crystal structure of HpaB in complex with FAD at 1.8 Å resolution opens up the possibility of the rational tuning of the substrate specificity and activity of this interesting class of phenol hydroxylases.

Exploiting Cofactor Versatility to Convert a FAD-Dependent Baeyer-Villiger Monooxygenase into a Ketoreductase.

Cyclohexanone monooxygenases (CHMOs) show very high catalytic specificity for natural Baeyer-Villiger (BV) reactions and promiscuous reduction reactions have not been reported to date. Wild-type CHMO from Acinetobacter sp. NCIMB 9871 was found to possess an innate, promiscuous ability to reduce an aromatic α-keto ester, but with poor yield and stereoselectivity. Structure-guided, site-directed mutagenesis drastically improved the catalytic carbonyl-reduction activity (yield up to 99 %) and stereoselectivity (ee up to 99 %), thereby converting this CHMO into a ketoreductase, which can reduce a range of differently substituted aromatic α-keto esters. The improved, promiscuous reduction activity of the mutant enzyme in comparison to the wild-type enzyme results from a decrease in the distance between the carbonyl moiety of the substrate and the hydrogen atom on N5 of the reduced flavin adenine dinucleotide (FAD) cofactor, as confirmed using docking and molecular dynamics simulations.

Straightforward Regeneration of Reduced Flavin Adenine Dinucleotide Required for Enzymatic Tryptophan Halogenation.

Flavin-dependent halogenases are known to regioselectively introduce halide substituents into aromatic moieties, for example, the indole ring of tryptophan. The process requires halide salts and oxygen instead of molecular halogen in the chemical halogenation. However, the reduced cofactor flavin adenine dinucleotide (FADH2) has to be regenerated using a flavin reductase. Consequently, coupled biocatalytic steps are usually applied for cofactor regeneration. Nicotinamide adenine dinucleotide (NADH) mimics can be employed stoichiometrically to replace enzymatic cofactor regeneration in biocatalytic halogenation. Chlorination of l-tryptophan is successfully performed using such NADH mimics. The efficiency of this approach has been compared to the previously established enzymatic regeneration system using the two auxiliary enzymes flavin reductase (PrnF) and alcohol dehydrogenase (ADH). The reaction rates of some of the tested mimics were found to exceed that of the enzymatic system. Continuous enzymatic halogenation reaction for reaction scale-up is also possible.

Lenalidomide Inhibits Thioredoxin Reductase (TrxR) in Multiple Myeloma (MM) Cells but Direct Inhibition of Trxr and Thioredoxin (Trx) Can Bypass Requirement of Cereblon (CRBN)

IntroductionA unique biological characteristic of MM plasma cells (PCs) is the substantial production of immunoglobulin proteins. This process requires disulfide bond formations that in turn generate large amounts of H2O2. The Trx-TrxR reductase system exists to combat this oxidative stress; however, cytotoxicity can occur when the cells' antioxidative capacity is overwhelmed. Recently, we demonstrated that IMiDs inhibit peroxidase-mediated decomposition of H2O2, and that lenalidomide sensitivity in MM correlates with the degree of antioxidative capacity (Sebastian et al. 2015 ASH). In this study we sought to better understand this inhibitory process.MethodsWe used various human MM cell lines with different sensitivity to lenalidomide. Cell viability was assessed by the MTT assay. Antioxidative capacity was measured via the total cellular oxidation of reduced flavin adenine dinucleotide (FADH2) and reduced nicotinamide adenine dinucleotide (NAD(P)H) after H2O2treatment. A CRBN CRISPR/Cas9 KO plasmid was used to generate CRBN knockout cells and lentivirus system was used to over-express CRBN in MM cells.ResultsWe found that IMiDs inhibit TrxR mediated intracellular H2O2 decomposition in a CRBN dependent fashion. Unlike lenalidomide, the direct TrxR inhibitor auranofin was very effective in inducing cell death in MM cell lines, in a CRBN independent fashion. By using MM.1R isogeneic cells with and without CRBN, and the CRBN-overexpressing OCIMY-5 cell line (transfected with wt-CRBN) we showed that auranofin treatment caused cytotoxicity irrespective of CRBN level of expression, while lenalidomide was only effective in CRBN expressing cells. Clinically achievable concentrations of auranofin treatment induced cell signals similar to lenalidomide, including degradation of IKZF1, IKZF3. As with lenalidomide, the sensitivity of CRBN expressing cell lines to auranofin correlated with their basal antioxidative capacity (MM.1S cells are sensitive and have with lower anti-oxidative capacity while RPMI-8226 is resistant due to its high anti-oxidative capacity).We also found that targeting Trx (downstream effector of TrxR) with the direct inhibitor PX12 overcame this cellular anti-oxidative capacity and inhibited cell proliferation in both RPMI-8226 and MM.1S in a CRBN independent fashion. Combination of auranofin with bortezomib showed a synergistic effect, but only in cells with lower anti-oxidative capacity. In contrast, PX-12, showed a synergistic effect with bortezomib in all analyzed MM cells lines, irrespective of their anti-oxidative capacity and CRBN expression.ConclusionsIMiDs inhibit TrxR-mediated intracellular H2O2decomposition in CRBN expressing MM. While the direct TrxR inhibitor, auranofin, bypassed the CRBN requirement for inducing cytotoxicity in MM, its cytotoxic effect could be overcome by the cells antioxidative capacity. Direct Trx inhibition (such as PX-12) seems like a potent strategy for MM therapy to overcome cellular CRBN requirements and higher anti-oxidative capacity cells. DisclosuresBergsagel:Novartis: Research Funding; Amgen, BMS, Novartis, Incyte: Consultancy. Stewart:celgene: Consultancy. Fonseca:Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Janssen: Consultancy; Bayer: Consultancy; Millennium, a Takeda Company: Consultancy; Millennium, a Takeda Company: Consultancy; BMS: Consultancy; Millennium, a Takeda Company: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Celgene: Consultancy; AMGEN: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Millennium, a Takeda Company: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Janssen: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Bayer: Consultancy; AMGEN: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Novartis: Consultancy; AMGEN: Consultancy; Sanofi: Consultancy; AMGEN: Consultancy; Sanofi: Consultancy; BMS: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms.

The putative flavin carrier family FlcA-C is important for Aspergillus fumigatus virulence

ABSTRACTAspergillus fumigatus is an opportunistic fungal pathogen and the most important species causing pulmonary fungal infections. The signaling by calcium is very important for A. fumigatus pathogenicity and it is regulated by the transcription factor CrzA. We have previously used used ChIP-seq (Chromatin Immunoprecipitation DNA sequencing) aiming to identify gene targets regulated by CrzA. We have identified among several genes regulated by calcium stress, the putative flavin transporter, flcA. This transporter belongs to a small protein family composed of FlcA, B, and C. The ΔflcA null mutant showed several phenotypes, such as morphological defects, increased sensitivity to calcium chelating-agent ethylene glycol tetraacetic acid (EGTA), cell wall or oxidative damaging agents and metals, repre-sentative of deficiencies in calcium signaling and iron homeostasis. Increasing calcium concentrations improved significantly the ΔflcA growth and conidiation, indicating that ΔflcA mutant has calcium insufficiency. Finally, ΔflcA-C mutants showed reduced flavin adenine dinucleotide (FAD) and were avirulent in a low dose murine infection model.

Functional Conversion of CPD and (6-4) Photolyases by Mutation.

Ultraviolet (UV) light from the sun damages DNA by forming a cyclobutane pyrimidine dimer (CPD) and pyrimidine(6-4)pyrimidone photoproducts [(6-4) PP]. Photolyase (PHR) enzymes utilize near-UV/blue light for DNA repair, which is initiated by light-induced electron transfer from the fully reduced flavin adenine dinucleotide chromophore. Despite similar structures and repair mechanisms, the functions of PHR are highly selective; CPD PHR repairs CPD, but not (6-4) PP, and vice versa. In this study, we attempted functional conversion between CPD and (6-4) PHRs. We found that a triple mutant of (6-4) PHR is able to repair the CPD photoproduct, though the repair efficiency is 1 order of magnitude lower than that of wild-type CPD PHR. Difference Fourier transform infrared spectra for repair demonstrate the lack of secondary structural alteration in the mutant, suggesting that the triple mutant gains substrate binding ability while it does not gain the optimized conformational changes from light-induced electron transfer to the release of the repaired DNA. Interestingly, the (6-4) photoproduct is not repaired by the reverse mutation of CPD PHR, and eight additional mutations (total of 11 mutations) introduced into CPD PHR are not sufficient. The observed asymmetric functional conversion is interpreted in terms of a more complex repair mechanism for (6-4) repair, which was supported by quantum chemical/molecular mechanical calculation. These results suggest that CPD PHR may represent an evolutionary origin for photolyase family proteins.

The Na+-Translocating NADH:Quinone Oxidoreductase Enhances Oxidative Stress in the Cytoplasm of Vibrio cholerae.

We searched for a source of reactive oxygen species (ROS) in the cytoplasm of the human pathogen Vibrio cholerae and addressed the mechanism of ROS formation using the dye 2',7'-dichlorofluorescein diacetate (DCFH-DA) in respiring cells. By comparing V. cholerae strains with or without active Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR), this respiratory sodium ion redox pump was identified as a producer of ROS in vivo The amount of cytoplasmic ROS detected in V. cholerae cells producing variants of Na(+)-NQR correlated well with rates of superoxide formation by the corresponding membrane fractions. Membranes from wild-type V. cholerae showed increased superoxide production activity (9.8 ± 0.6 μmol superoxide min(-1) mg(-1) membrane protein) compared to membranes from the mutant lacking Na(+)-NQR (0.18 ± 0.01 μmol min(-1) mg(-1)). Overexpression of plasmid-encoded Na(+)-NQR in the nqr deletion strain resulted in a drastic increase in the formation of superoxide (42.6 ± 2.8 μmol min(-1) mg(-1)). By analyzing a variant of Na(+)-NQR devoid of quinone reduction activity, we identified the reduced flavin adenine dinucleotide (FAD) cofactor of cytoplasmic NqrF subunit as the site for intracellular superoxide formation in V. cholerae The impact of superoxide formation by the Na(+)-NQR on the virulence of V. cholerae is discussed. In several studies, it was demonstrated that the Na(+)-NQR in V. cholerae affects virulence in a yet unknown manner. We identified the reduced FAD cofactor in the NADH-oxidizing NqrF subunit of the Na(+)-NQR as the site of superoxide formation in the cytoplasm of V. cholerae Our study provides the framework to understand how reactive oxygen species formed during respiration could participate in the regulated expression of virulence factors during the transition from aerobic to microaerophilic (intestinal) habitats. This hypothesis may turn out to be right for many other pathogens which, like V. cholerae, depend on the Na(+)-NQR as the sole electrogenic NADH dehydrogenase.