Identification of Up- and Down-Regulated Proteins in Pemetrexed-Resistant Human Lung Adenocarcinoma: Flavin Reductase and Calreticulin Play Key Roles in the Development of Pemetrexed-Associated Resistance.

Drug resistance is one of the major causes of cancer chemotherapy failure. In the current study, we used a pair of lung adenocarcinoma cell lines, A549 and the pemetrexed-resistant A549/PEM cells, as a model to monitor resistance-dependent cellular responses and identify potential therapeutic targets. By means of 2D differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), we investigated the global protein expression alterations induced by pemetrexed treatment and resistance. The proteomic result revealed that pemetrexed exposure obviously altered the expression of 81 proteins in the A549 cells, whereas no significant response was observed in the similarly treated A549/PEM cells, hence implying an association between these proteins and the drug-specific response. Moreover, 72 proteins including flavin reductase and calreticulin demonstrated differential expression between the A549 and A549/PEM cells, indicating baseline resistance. Additional tests employed siRNA silencing, protein overexpression, cell viability analysis, and analysis of apoptosis to examine and confirm the potency of flavin reductase and calreticulin proteins in the development of pemetrexed resistance. In summary, by using a proteomic approach, we identified numerous proteins, including flavin reductase and calreticulin, involved in pemetrexed drug resistance-developing mechanisms. Our results provide useful diagnostic markers and therapeutic candidates for pemetrexed-resistant lung cancer treatment.

H(2)O(2) production in species of the Lactobacillus acidophilus group: a central role for a novel NADH-dependent flavin reductase.

Hydrogen peroxide production is a well-known trait of many bacterial species associated with the human body. In the presence of oxygen, the probiotic lactic acid bacterium Lactobacillus johnsonii NCC 533 excretes up to 1 mM H(2)O(2), inducing growth stagnation and cell death. Disruption of genes commonly assumed to be involved in H(2)O(2) production (e.g., pyruvate oxidase, NADH oxidase, and lactate oxidase) did not affect this. Here we describe the purification of a novel NADH-dependent flavin reductase encoded by two highly similar genes (LJ_0548 and LJ_0549) that are conserved in lactobacilli belonging to the Lactobacillus acidophilus group. The genes are predicted to encode two 20-kDa proteins containing flavin mononucleotide (FMN) reductase conserved domains. Reductase activity requires FMN, flavin adenine dinucleotide (FAD), or riboflavin and is specific for NADH and not NADPH. The Km for FMN is 30 ± 8 µM, in accordance with its proposed in vivo role in H(2)O(2) production. Deletion of the encoding genes in L. johnsonii led to a 40-fold reduction of hydrogen peroxide formation. H(2)O(2) production in this mutant could only be restored by in trans complementation of both genes. Our work identifies a novel, conserved NADH-dependent flavin reductase that is prominently involved in H(2)O(2) production in L. johnsonii.

A Rhizobium selenitireducens protein showing selenite reductase activity.

Biobarriers remove, via precipitation, the metalloid selenite (SeO3⁻²) from groundwater; a process that involves the biological reduction of soluble SeO3⁻² to insoluble elemental red selenium (Se°). The enzymes associated with this reduction process are poorly understood. In Rhizobium selenitireducens at least two enzymes are potentially involved; one, a nitrite reductase reduces SeO3⁻² to Se° but another protein may also be involved which is investigated in this study. Proteins from R. selenitireducens cells were precipitated with ammonium sulfate and run on native electrophoresis gels. When these gels were incubated with NADH and SeO3⁻² a band of precipitated Se° developed signifying the presence of a SeO3⁻² reducing protein. Bands were cut from the gel and analyzed for peptides via LCMSMS. The amino acid sequences associated with the bands indicated the presence of an NADH:flavin oxidoreductase that resembles YP_001326930 from Sinorhizobium medicae. The protein is part of a protein family termed old-yellow-enzymes (OYE) that contain a flavin binding domain. OYE enzymes are often involved in protecting cells from oxidative stress and, due in part to an active site that has a highly accessible binding pocket, are generally active on a wide range of substrates. This report is the first of an OYE enzyme being involved in SeO3⁻² reduction.

Camphor pathway redux: functional recombinant expression of 2,5- and 3,6-diketocamphane monooxygenases of Pseudomonas putida ATCC 17453 with their cognate flavin reductase catalyzing Baeyer-Villiger reactions.

Whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-DKCMO and 3,6-DKCMO, respectively) in the initial catabolic steps of (+) and (-) isomeric forms of camphor (CAM) metabolism in Pseudomonas putida ATCC 17453 are relatively well characterized, the actual identity of the flavin reductase (Fred) component that provides the reduced flavin to the oxygenases has hitherto been ill defined. In this study, a 37-kDa Fred was purified from a camphor-induced culture of P. putida ATCC 17453 and this facilitated cloning and characterization of the requisite protein. The active Fred is a homodimer with a subunit molecular weight of 18,000 that uses NADH as an electron donor (Km = 32 µM), and it catalyzes the reduction of flavin mononucleotide (FMN) (Km = 3.6 µM; kcat = 283 s(-1)) in preference to flavin adenine dinucleotide (FAD) (Km = 19 µM; kcat = 128 s(-1)). Sequence determination of ∼40 kb of the CAM degradation plasmid revealed the locations of two isofunctional 2,5-DKCMO genes (camE25-1 for 2,5-DKCMO-1 and camE25-2 for 2,5-DKCMO-2) as well as that of a 3,6-DKCMO-encoding gene (camE36). In addition, by pulsed-field gel electrophoresis, the CAM plasmid was established to be linear and ∼533 kb in length. To enable functional assessment of the two-component monooxygenase system in Baeyer-Villiger oxidations, recombinant plasmids expressing Fred in tandem with the respective 2,5-DKCMO- and 3,6-DKCMO-encoding genes in Escherichia coli were constructed. Comparative substrate profiling of the isofunctional 2,5-DCKMOs did not yield obvious differences in Baeyer-Villiger biooxidations, but they are distinct from 3,6-DKCMO in the stereoselective oxygenations with various mono- and bicyclic ketone substrates.

High pressure refolding, purification, and crystallization of flavin reductase from Sulfolobus tokodaii strain 7.

Flavin reductase HpaC(St) catalyzes the reduction of free flavins using NADH or NADPH. High hydrostatic pressure was used for the solubilization and refolding of HpaC(St), which was expressed as inclusion bodies in Escherichia coli to achieve high yield in a flavin-free form. The refolded HpaC(St) was purified using Ni-affinity chromatography followed by a heat treatment, which gave a single band on SDS-PAGE. The purified refolded HpaC(St) did not contain FMN, unlike the same enzyme expressed as a soluble protein. After the addition of FMN to the protein solution, the refolded enzyme showed a higher activity than the enzyme expressed as the soluble protein. Crystals of the refolded enzyme were obtained by adding FMN, FAD, or riboflavin to the protein solution and without the addition of flavin compound.

Structural and biochemical characterization of an extremely thermostable FMN-dependent NADH-indigo reductase from Bacillus smithii.

The FMN-dependent NADH-indigo reductase gene from the thermophilic bacterium Bacillus smithii was overexpressed in Escherichia coli. The expressed enzyme functioned as a highly thermostable indigo reductase that retained complete activity even after incubation at 100 °C for 10 min. Furthermore, B. smithii indigo reductase exhibited high stability over a wider pH range and longer storage periods compared with indigo reductases previously identified from other sources. The enzyme catalyzed the reduction of various azo compounds and indigo carmine. The crystal structures of the wild-type enzyme in complex with FMN/N-cyclohexyl-2-aminoethanesulfonate (CHES) and the Y151F mutant enzyme in complex with FMN were determined by the molecular replacement method and refined at resolutions of 1.97 and 1.95 Å, respectively. Then, indigo carmine molecule was modeled into the active site using the molecular docking simulation and the binding mode of indigo carmine was elucidated. In addition, the structure of B. cohnii indigo reductase, which is relatively less stable than B. smithii indigo reductase, was constructed by homology modeling. The factor contributing to the considerably higher thermostability of B. smithii indigo reductase was analyzed by comparing its structure with that of B. cohnii indigo reductase, which revealed that intersubunit aromatic interactions (F105-F172' and F172-F105') may be responsible for the high thermostability of B. smithii indigo reductase. Notably, site-directed mutagenesis results showed that F105 plays a major role in the intersubunit aromatic interaction.

Dissimilatory iron reduction in Escherichia coli: identification of CymA of Shewanella oneidensis and NapC of E. coli as ferric reductases.

Over geological time scales, microbial reduction of chelated Fe(III) or Fe(III) minerals has profoundly affected today's composition of our bio- and geosphere. However, the electron transfer reactions that are specific and defining for dissimilatory iron(III)-reducing (DIR) bacteria are not well understood. Using a synthetic biology approach involving the reconstruction of the putative electron transport chain of the DIR bacterium Shewanella oneidensis MR-1 in Escherichia coli, we showed that expression of cymA was necessary and sufficient to convert E. coli into a DIR bacterium. In intact cells, the Fe(III)-reducing activity was limited to Fe(III) NTA as electron acceptor. In vitro biochemical analysis indicated that CymA, which is a cytoplasmic membrane-associated tetrahaem c-type cytochrome, carries reductase activity towards Fe(III) NTA, Fe(III) citrate, as well as to AQDS, a humic acid analogue. The in vitro specific activities of Fe(III) citrate reductase and AQDS reductase of E. coli spheroplasts were 10x and 30x higher, respectively, relative to the specific rates observed in intact cells, suggesting that access of chelated and insoluble forms of Fe(III) and AQDS is restricted in whole cells. Interestingly, the E. coli CymA orthologue NapC also carried ferric reductase activity. Our data support the argument that the biochemical mechanism of Fe(III) reduction per se was not the key innovation leading to environmental relevant DIR bacteria. Rather, the evolution of an extension of the electron transfer pathway from the Fe(III) reductase CymA to the cell surface via a system of periplasmic and outer membrane cytochrome proteins enabled access to diffusion-impaired electron acceptors.

Alternative NAD(P)H dehydrogenases of plant mitochondria.

Plant mitochondria have a highly branched electron transport chain that provides great flexibility for oxidation of cytosolic and matrix NAD(P)H. In addition to the universal electron transport chain found in many organisms, plants have alternative NAD(P)H dehydrogenases in the first part of the chain and a second oxidase, the alternative oxidase, in the latter part. The alternative activities are nonproton pumping and allow for NAD(P)H oxidation with varying levels of energy conservation. This provides a mechanism for plants to, for example, remove excess reducing power and balance the redox poise of the cell. This review presents our current understanding of the alternative NAD(P)H dehydrogenases present in plant mitochondria.

Lot6p from Saccharomyces cerevisiae is a FMN-dependent reductase with a potential role in quinone detoxification.

NAD(P)H:quinone acceptor oxidoreductases are flavoenzymes expressed in the cytoplasm of many tissues and afford protection against the cytotoxic effects of electrophilic quinones by catalyzing a strict two-electron reduction. Such enzymes have been reported from several mammalian sources, e.g. human, mouse and rat, and from plant species. Here, we report identification of Lot6p (YLR011wp), the first soluble quinone reductase from the unicellular model organism Saccharomyces cerevisiae. Localization studies using an antibody raised against Lot6p as well as microscopic inspection of Lot6p-GFP demonstrated accumulation of the enzyme in the cytosol of yeast cells. Despite sharing only 23% similarity to type 1 human quinone reductase, Lot6p possesses biochemical properties that are similar to its human counterpart. The enzyme catalyzes a two-electron reduction of a series of natural and artificial quinone substrates at the expense of either NADH or NADPH. The kinetic mechanism follows a ping-pong bi-bi reaction scheme, with K(M) values of 1.6-11 microm for various quinones. Dicoumarol and Cibacron Marine, two well-known inhibitors of the quinone reductase family, bind to Lot6p and inhibit its activity. In vivo experiments demonstrate that the enzymatic activity of Lot6p is consistent with the phenotype of both Deltalot6 and Lot6p overexpressing strains, suggesting that Lot6p may play a role in managing oxidative stress in yeast.

Isolation of a soluble and membrane-associated Fe(III) reductase from the thermophile, Thermus scotoductus (SA-01).

The Fe(III) reductase activity was studied in the South African Fe(III)-reducing bacterium, Thermus scotoductus (SA-01). Fractionation studies revealed that the membrane as well as the soluble fraction contained NAD(P)H-dependent Fe(III) reductase activity. The membrane-associated enzyme was solubilized by KCl treatment and purified to electrophoretic homogeneity by hydrophobic interaction chromatography. A combination of ion-exchange and gel filtration chromatography was used to purify the soluble enzyme to apparent homogeneity. The molecular mass of the membrane-associated Fe(III) reductase was estimated to be 49 kDa, whereas the soluble Fe(III) reductase had an apparent molecular mass of 37 kDa. Optimum activity for the membrane-associated enzyme was observed at around 75 degrees C, whereas the soluble enzyme exhibited a temperature optimum at 60 degrees C.

Purification, crystallization and preliminary X-ray diffraction analysis of ArsH from Synechocystis sp. strain PCC 6803.

ArsH is an NADPH-dependent flavin mononucleotide reductase and is frequently encoded as part of an ars operon. The function of the arsH gene remains to be characterized. Crystallization and structural studies may contribute to elucidating the specific biological function of ArsH associated with arsenic resistance. ArsH from Synechocystis sp. strain PCC 6803 was overproduced, purified and crystallized. Crystals were obtained by the sitting-drop vapour-diffusion method. Diffraction data were collected and processed to a resolution of 1.6 Å. The crystals belonged to the tetragonal space group I4122, with unit-cell parameters a = b = 127.94, c = 65.86 Šand one molecule in the asymmetric unit. Size-exclusion chromatography and molecular-replacement results showed that the ArsH formed a tetramer. Further structural analysis and comparison with ArsH from Sinorhizobium meliloti will provide information about the oligomerization of ArsH.

A novel ferric reductase purified from Magnetospirillum gryphiswaldense MSR-1.

A ferric reductase was purified into an electrophoretically homologous state from Magnetospirillum gryphiswaldense MSR-1 strain. The enzyme was found within the cytoplasm and associated with the cytoplasmic membrane. The molecular weight of the purified enzyme was calculated as 16.1 kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was almost identical to that calibrated using agarose gel filtration chromatography. It was NADH-dependent and required flavin mononucleotide as a cofactor. The optimal reaction temperature and pH values were 30 degrees C and 6.5, respectively. The K(m) and Vmax values for ferric citrate were 45.1 microM: and 1.216 microM: min(-1), respectively. Though ferric reductase activity could be inhibited by Co(2+), Cu(2+), Mn(2+), and Zn(2+), even high concentrations of Mg(2+) ions have failed to accomplish such enzyme inhibition. Furthermore, the molecular weight, the N-terminal sequence, and the activity of ferric reductase from MSR-1 are not matching with the enzyme preparation obtained from an analogous strain M. magnetotacticum (MS-1). Therefore, it is concluded that the ferric reductase of M. grysphiwaldense and M. magnetotacticum strains are two different enzymes.

Butane monooxygenase of 'Pseudomonas butanovora': purification and biochemical characterization of a terminal-alkane hydroxylating diiron monooxygenase.

Butane monooxygenase (sBMO) has been purified to homogeneity from the Gram-negative beta-proteobacterium 'Pseudomonas butanovora' and confirmed to be a three-component diiron monooxygenase system. The reconstituted enzyme complex oxidized C(3)-C(6) linear and branched aliphatic alkanes, which are growth substrates for 'P. butanovora'. The sBMO complex was composed of an iron-containing hydroxylase (BMOH), a flavo-iron sulfur-containing NADH-oxidoreductase (BMOR) and a small regulatory component protein (BMOB). The physical characteristics of sBMO were remarkably similar to the sMMO family of soluble multicomponent diiron monooxgenases. However, the catalytic properties of sBMO were quantitatively different in regard to inactivation in the presence of substrate and product distribution. BMOH was capable of ethene oxidation when supplied with H(2)O(2) and ethene (known as the peroxide shunt), but this activity was at least three orders of magnitude less than that observed for the hydroxylase of sMMO of Methylosinus trichosporium OB3b. BMOH and BMOR were efficient in the oxidation of ethene in the absence of BMOB with regard to rate of reaction and product yield. Regiospecificity of sBMO was strongly biased towards primary hydroxylation, with > or = 80 % of the hydroxylations occurring at the terminal carbon atom.

Characterization of a thermostable NADPH:FMN oxidoreductase from the mesophilic bacterium Bacillus subtilis.

The gene yhdA from Bacillus subtilis encoding a putative flavin mononucleotide (FMN)-dependent oxidoreductase was cloned and heterologously expressed in Escherichia coli. The purified enzyme has a noncovalently bound FMN cofactor, which is preferentially reduced by NADPH, indicating that YhdA is a NADPH:FMN oxidoreductase. The rate of NADPH oxidation is enhanced by the addition of external FMN, and analysis of initial rate measurements reveals the occurrence of a ternary complex in a bi-bi reaction mechanism. YhdA has also been shown to reductively cleave the -N=N- bond in azo dyes at the expense of NADPH, and hence, it possesses azoreductase activity, however, at a rate 100 times slower than that found for FMN. Using Cibacron Marine as a model compound, we could demonstrate that the dye is a competitive inhibitor of NADPH and FMN. The utilization of NADPH and the absence of a flavin semiquinone radical distinguish YhdA from flavodoxins, which adopt the same structural fold, i.e., a five-stranded beta sheet sandwiched by five alpha helices. The native molecular-mass of YhdA was determined to be 76 kDa, suggesting that the protein occurs as a tetramer, whereas the YhdA homologue in Saccharomyces cerevisiae (YLR011wp) forms a dimer in solution. Interestingly, the different oligomerization of these homologous proteins correlates to their thermostability, with YhdA exhibiting a melting point of 86.5 degrees C, which is 26.3 degrees C higher than that for the yeast protein. This unusually high melting point is proposed to be the result of increased hydrophobic packing between dimers and the additional presence of four salt bridges stabilizing the dimer-dimer interface.

Iodotyrosine deiodinase is the first mammalian member of the NADH oxidase/flavin reductase superfamily.

The enzyme responsible for iodide salvage in the thyroid, iodotyrosine deiodinase, was solubilized from porcine thyroid microsomes by limited proteolysis with trypsin. The resulting protein retained deiodinase activity and was purified using anion exchange, dye, and hydrophobic chromatography successively. Peptide sequencing of the final isolate identified the gene responsible for the deiodinase. The amino acid sequence of the porcine enzyme is highly homologous to corresponding genes in a variety of mammals including humans, and the mouse gene was expressed in human embryonic kidney 293 cells to confirm its identity. The amino acid sequence of the deiodinase suggests the presence of three domains. The N-terminal domain provides a membrane anchor. The intermediate domain contains the highest sequence variability and lacks homology to structural motifs available in the common databases. The C-terminal domain is highly conserved and resembles bacterial enzymes of the NADH oxidase/flavin reductase superfamily. A three-dimensional model of the deiodinase based on the coordinates of the minor nitroreductase of Escherichia coli indicates that a Cys common to all of the mammal sequences is located adjacent to bound FMN. However, the deiodinase is not structurally related to other known flavoproteins containing redox-active cysteines or the iodothyronine deiodinases containing an active site selenocysteine.

Isolation and biochemical characterization of two soluble iron(III) reductases from Paracoccus denitrificans.

Two soluble enzymes (FerA and FerB) catalyzing the reduction of a number of iron(III) complexes by NADH, were purified to near homogeneity from the aerobically grown iron-limited culture of Paracoccus denitrificans using a combination of anion-exchange chromatography (Sepharose Q), chromatofocusing (Mono P), and gel permeation chromatography (Superose 12). FerA is a monomer with a molecular mass of 19 kDa, whereas FerB exhibited a molecular mass of about 55 kDa and consists of probably two identical subunits. FerA can be classified as an NADH:flavin oxidoreductase with a sequential reaction mechanism. It requires the addition of FMN or riboflavin for activity on Fe(III) substrates. In these reactions, the apparent substrate specificity of FerA seems to stem exclusively from different chemical reactivities of Fe(III) compounds with the free reduced flavin produced by the enzyme. Observations on reducibility of Fe(III) chelated by vicinal dihydroxy ligands support the view that FerA takes part in releasing iron from the catechol type siderophores synthesized by P. denitrificans. Contrary to FerA, the purified FerB contains a noncovalently bound redox-active FAD coenzyme, can utilize NADPH in place of NADH, does not reduce free FMN at an appreciable rate, and gives a ping-pong type kinetic pattern with NADH and Fe(III)-nitrilotriacetate as substrates. FerB is able to reduce chromate, in agreement with the fact that its N-terminus bears a homology to the previously described chromate reductase from Pseudomonas putida. Besides this, it also readily reduces quinones like ubiquinone-0 (Q0) or unsubstituted p-benzoquinone.

Flavin reductase coupling with two monooxygenases involved in dibenzothiophene desulfurization: purification and characterization from a non-desulfurizing bacterium, Paenibacillus polymyxa A-1.

The dibenzothiophene (DBT) desulfurizing bacterium metabolizes DBT to form 2-hydroxybiphenyl without breaking the carbon skeleton. Of the DBT desulfurization enzymes, DszC and DszA catalyze monooxygenation reactions, both requiring flavin reductase. We searched for non-DBT-desulfurizing microorganisms producing a flavin reductase that couples more efficiently with DszC than that produced by the DBT desulfurizing bacterium Rhodococcus erythropolis D-1, and found Paenibacillus polymyxa A-1 to be a promising strain. The enzyme was purified to complete homogeneity. K(m) values for FMN and NADH were 2.1 microM and 0.57 mM, respectively. Flavin compounds were good substrates, some nitroaromatic compounds were also active, and regarding the electron donor, the activity for NADPH was about 1.5 times that for NADH. In the coupling assay with DszC, only FMN or riboflavin acted as the electron acceptor. The coupling reactions of P. polymyxa A-1 flavin reductase with DszC and DszA proceeded more efficiently (3.5- and 5-fold, respectively) than those of R. erythropolis D-1 flavin reductase when identical enzyme activities of each flavin reductase were added to the reaction mixture. The result of the coupling reaction suggested that, in the microbial DBT desulfurization, flavin reductase from the non-DBT-desulfurizing bacterium was superior to that from the DBT-desulfurizing bacterium.

Biochemical characterization of StyAB from Pseudomonas sp. strain VLB120 as a two-component flavin-diffusible monooxygenase.

Pseudomonas sp. VLB120 uses styrene as a sole source of carbon and energy. The first step in this metabolic pathway is catalyzed by an oxygenase (StyA) and a NADH-flavin oxidoreductase (StyB). Both components have been isolated from wild-type Pseudomonas strain VLB120 as well as from recombinant Escherichia coli. StyA from both sources is a dimer, with a subunit size of 47 kDa, and catalyzes the enantioselective epoxidation of CC double bonds. Styrene is exclusively converted to S-styrene oxide with a specific activity of 2.1 U mg(-1) (k(cat) = 1.6 s(-1)) and K(m) values for styrene of 0.45 +/- 0.05 mM (wild type) and 0.38 +/- 0.09 mM (recombinant). The epoxidation reaction depends on the presence of a NADH-flavin adenine dinucleotide (NADH-FAD) oxidoreductase for the supply of reduced FAD. StyB is a dimer with a molecular mass of 18 kDa and a NADH oxidation activity of 200 U mg(-1) (k(cat) [NADH] = 60 s(-1)). Steady-state kinetics determined for StyB indicate a mechanism of sequential binding of NADH and flavin to StyB. This enzyme reduces FAD as well as flavin mononucleotide and riboflavin. The NADH oxidation activity does not depend on the presence of StyA. During the epoxidation reaction, no formation of a complex of StyA and StyB has been observed, suggesting that electron transport between reductase and oxygenase occurs via a diffusing flavin.