For Flavin Adenine Dinucleotide Research Articles

Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

Essential for reactive oxygen species (EROS) protein is a recently identified molecular chaperone of NOX2 (gp91phox), the catalytic subunit of phagocyte NADPH oxidase. Deficiency in EROS is a recently identified cause for chronic granulomatous disease, a genetic disorder with recurrent bacterial and fungal infections. Here, we report a cryo-EM structure of the EROS-NOX2-p22phox heterotrimeric complex at an overall resolution of 3.56Å. EROS and p22phox are situated on the opposite sides of NOX2, and there is no direct contact between them. EROS associates with NOX2 through two antiparallel transmembrane (TM) α-helices and multiple β-strands that form hydrogen bonds with the cytoplasmic domain of NOX2. EROS binding induces a 79° upward bend of TM2 and a 48° backward rotation of the lower part of TM6 in NOX2, resulting in an increase in the distance between the two hemes and a shift of the binding site for flavin adenine dinucleotide (FAD). These conformational changes are expected to compromise superoxide production by NOX2, suggesting that the EROS-bound NOX2 is in a protected state against activation. Phorbol myristate acetate, an activator of NOX2 in vitro, is able to induce dissociation of NOX2 from EROS with concurrent increase in FAD binding and superoxide production in a transfected COS-7 model. In differentiated neutrophil-like HL-60, the majority of NOX2 on the cell surface is dissociated with EROS. Further studies are required to delineate how EROS dissociates from NOX2 during its transport to cell surface, which may be a potential mechanism for regulation of NOX2 activation.

Effect of Fermentation Scale on Microbiota Dynamics and Metabolic Functions for Indigo Reduction.

During indigo dyeing fermentation, indigo reduction for the solubilization of indigo particles occurs through the action of microbiota under anaerobic alkaline conditions. The original microbiota in the raw material (sukumo: composted indigo plant) should be appropriately converged toward the extracellular electron transfer (EET)-occurring microbiota by adjusting environmental factors for indigo reduction. The convergence mechanisms of microbiota, microbial physiological basis for indigo reduction, and microbiota led by different velocities in the decrease in redox potential (ORP) at different fermentation scales were analyzed. A rapid ORP decrease was realized in the big batch, excluding Actinomycetota effectively and dominating Alkalibacterium, which largely contributed to the effective indigo reduction. Functional analyses of the microbiota related to strong indigo reduction on approximately day 30 indicated that the carbohydrate metabolism, prokaryotic defense system, and gene regulatory functions are important. Because the major constituent in the big batch was Alkalibacterium pelagium, we attempted to identify genes related to EET in its genome. Each set of genes for flavin adenine dinucleotide (FAD) transportation to modify the flavin mononucleotide (FMN)-associated family, electron transfer from NADH to the FMN-associated family, and demethylmenaquinone (DMK) synthesis were identified in the genome sequence. The correlation between indigo intensity reduction and metabolic functions suggests that V/A-type H+/Na+-transporting ATPase and NAD(P)H-producing enzymes drive membrane transportations and energization in the EET system, respectively.

Magnetic molecularly imprinted polymers for selectively adsorbing flavins and their effects on bioremoval of Acid Red 18 and Cr(VI)

AbstractBACKGROUNDFlavins can be used as redox mediators to accelerate anaerobic biotransformation of refractory pollutants. For better application of flavins, suitable immobilization methods need to be developed. As most microorganisms can synthesize flavins, it is a good strategy to develop selective solid sorbents for immobilizing the flavins released by waste biomass including waste activated sludge (WAS).RESULTSNovel core–shell magnetic molecularly imprinted polymers (MMIPs) for flavins were successfully prepared. The adsorption amount of 10 mg of MMIPs for flavin mononucleotide (FMN) could reach 29.19 μmol g−1, which was around 24.9‐fold higher than that of magnetic non‐molecularly imprinted polymers (1.17 μmol g−1). Their isothermal adsorption process followed the Langmuir model. Similar adsorption behaviors of MMIPs were also observed for flavin adenine dinucleotide (FAD) and riboflavin. The three flavins adsorbed on MMIPs enhanced the removal rates of azo dye Acid Red 18 (AR 18) and Cr(VI) by Shewanella oneidensis MR‐1. Moreover, the catalytic performance of MMIPs–FMN could remain at 97.6% of the original value after five repeated usages. Further studies found that MMIPs could efficiently adsorb FAD from the lysate of WAS, and MMIPs–FAD enhanced the decolorization of AR 18.CONCLUSIONSThe novel MMIPs could selectively adsorb flavins. MMIPs–flavins exhibited good catalytic and repeated usage performances, indicating that they could be used for accelerating anaerobic biotransformation of refractory pollutants. © 2022 Society of Chemical Industry (SCI).

Structural insights of a cellobiose dehydrogenase enzyme from the basidiomycetes fungus Termitomyces clypeatus

Filamentous fungi secrete various oxidative enzymes to degrade the glycosidic bonds of polysaccharides. Cellobiose dehydrogenase (CDH) (E.C.1.1.99.18) is one of the important lignocellulose degrading enzymes produced by various filamentous fungi. It contains two stereo specific ligand binding domains, cytochrome and dehydrogenase - one for heme and the other for flavin adenine dinucleotide (FAD) respectively. The enzyme is of commercial importance for its use in amperometric biosensor, biofuel production, lactose determination in food, bioremediation etc. Termitomyces clypeatus, an edible fungus belonging to the basidiomycetes group, is a good producer of CDH. In this paper we have analyzed the structural properties of this enzyme from T. clypeatus and identified a distinct carbohydrate binding module (CBM) which is not present in most fungi belonging to the basidiomycetes group. In addition, the dehydrogenase domain of T. clypeatus CDH exhibited the absence of cellulose binding residues which is in contrast to the dehydrogenase domains of CDH of other basidiomycetes. Sequence analysis of cytochrome domain showed that the important residues of this domain were conserved like in other fungal CDHs. Phylogenetic tree, constructed using basidiomycetes and ascomycetes CDH sequences, has shown that very surprisingly the CDH from T. clypeatus, which is classified as a basidiomycetes fungus, is clustered with the ascomycetes group. A homology model of this protein has been constructed using the CDH enzyme of ascomycetes fungus Myricoccum thermophilum as a template since it has been found to be the best match sequence with T. clypeatus CDH. We also have modelled the protein with its substrate, cellobiose, which has helped us to identify the substrate interacting residues (L354, P606, T629, R631, Y649, N732, H733 and N781) localized within its dehydrogenase domain. Our computational investigation revealed for the first time the presence of all three domains - cytochrome, dehydrogenase and CBM - in the CDH of T. clypeatus, a basidiomycetes fungus. In addition to discovering the unique structural attributes of this enzyme from T. clypeatus, our study also discusses the possible phylogenetic status of this fungus.

A novel electrochemical biosensor based on TetX2 monooxygenase immobilized on a nano-porous glassy carbon electrode for tetracycline residue detection

Different carbon-based nanostructures were used to investigate direct electron transfer (DET) of TetX2 monooxygenase (TetX2), and an enzyme-based biosensor for sensitive determination of tetracycline (TC) also fabricated. A polyethyleneimine (PEI) with positive charge groups was used for immobilization of TetX2 on modified glassy carbon electrodes. Cyclic voltammetry (CV) was employed to study the electrochemical characteristics of the immobilized enzyme and the performance of the proposed biosensor. Amongst multiple carbon-modified electrodes, nano-porous glassy carbon electrode (NPGCE) was selected because of its amplified signal response for flavin adenine dinucleotide (FAD) and superior electrocatalytic behavior toward oxygen reduction. The cyclic voltammogram of PEI/TetX2/NPGCE showed two couple of well-defined and quasi-reversible redox peaks of FAD, consistent with the realization of DET. The prepared electrode was then successfully introduced as a biosensing interface based on the oxygen reduction peak current, resulting in a linear range response from 0.5 to 5 μM with a good detection limit of 18 nM. The as-fabricated electrode demonstrates a fast response and excellent stability for the detection of TC. The results indicate that this simple, rapid, eco-friendly and economic strategy of PEI/TetX2/NPGCE preparation has potential for the fabrication of an enzyme-based biosensor for the practical detection of TC in food products.

Free flavins accelerate release of ferrous iron from iron storage proteins by both free flavin-dependent and -independent ferric reductases in Escherichia coli.

Ferredoxin NADP+ oxidoreductase (Fpr) and oxygen-insensitive NAD(P)H nitroreductase (NfnB) are purified from Escherichia coli JM109 (E. coli JM109) as a predominant free flavin-independent ferric reductase. In the present study, we prepared natural iron storage proteins, E. coli ferritin A (FtnA) and bacterioferritin (Bfr), to show the effective ferrous iron release from these proteins by Fpr and NfnB in the presence of free flavins. Fpr and NfnB showed flavin reductase activity for flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and riboflavin, and their ferrous iron release activities were positively associated with the catalytic efficiencies (kcat/Km) for individual flavins. The ferrous iron release activity of E. coli cell-free extracts was affected by flavin reductase activity of the extracts. The Butyl TOYOPEARL column chromatography of the extracts, on the basis of NAD(P)H-dependent flavin reductase activity, resulted in the separation of six active fractions containing Fpr, NfnB, NAD(P)H-quinone oxidoreductase (QOR), flavin reductase (Fre) or alkyl hydroperoxide reductase subunit F (AhpF) as major components. Like Fpr and NfnB, recombinant QOR, Fre, and AhpF showed flavin reductase activity and ferrous iron release activity in the presence of free flavins, indicating an association of flavin reductase activity with ferrous iron releasing activity. Taken together, both free flavin-dependent and free flavin-independent ferric reductases in E. coli require free flavins to mediate an electron transfer from NAD(P)H to ferric iron in the iron storage proteins for the effective ferrous iron release.

Crystal Structure of the Apo-Form of NADPH-Dependent Thioredoxin Reductase from a Methane-Producing Archaeon.

The redox regulation of proteins via reversible dithiol/disulfide exchange reactions involves the thioredoxin system, which is composed of a reductant, a thioredoxin reductase (TR), and thioredoxin (Trx). In the pyridine nucleotide-dependent Trx reduction pathway, reducing equivalents, typically from reduced nicotinamide adenine dinucleotide phosphate (NADPH), are transferred from NADPH-TR (NTR) to Trx and, in turn, to target proteins, thus resulting in the reversible modification of the structural and functional properties of the targets. NTR enzymes contain three functional sites: an NADPH binding pocket, a non-covalently bound flavin cofactor, and a redox-active disulfide in the form of CxxC. With the aim of increasing our knowledge of the thioredoxin system in archaea, we here report the high-resolution crystal structure of NTR from the methane-generating organism Methanosarcina mazei strain Gö1 (MmNTR) at 2.6 Å resolution. Based on the crystals presently described, MmNTR assumes an overall fold that is nearly identical to the archetypal fold of authentic NTRs; however, surprisingly, we observed no electron density for flavin adenine dinucleotide (FAD) despite the well-defined and conserved FAD-binding cavity in the folded module. Remarkably, the dimers of the apo-protein within the crystal were different from those observed by small angle X-ray scattering (SAXS) for the holo-protein, suggesting that the binding of the flavin cofactor does not require major protein structural rearrangements. Rather, binding results in the stabilization of essential parts of the structure, such as those involved in dimer stabilization. Altogether, this structure represents the example of an apo-form of an NTR that yields important insight into the effects of the cofactor on protein folding.

Molecular Cloning and Functional Characterization of the Dual Oxidase (BmDuox) Gene from the Silkworm Bombyx mori

Reactive oxygen species (ROS) from nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and their related dual oxidases are known to have significant roles in innate immunity and cell proliferation. In this study, the 5,545 bp cDNA of the silkworm Bombyx mori dual oxidase (BmDuox) gene containing a full-length open reading frame was cloned. It was shown to include an N-terminal signal peptide consisting of 28 amino acid residues, a 240 bp 5′-terminal untranslated region (5′-UTR), an 802 bp 3′-terminal region (3′-UTR), which contains nine ATTTA motifs, and a 4,503 bp open reading frame encoding a polypeptide of 1,500 amino acid residues. Structural analysis indicated that BmDuox contains a typical peroxidase domain at the N-terminus followed by a calcium-binding domain, a ferric-reducing domain, six transmembrane regions and binding domains for flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD). Transcriptional analysis revealed that BmDuox mRNA was expressed more highly in the head, testis and trachea compared to the midgut, hemocyte, Malpighian tube, ovary, fat bodies and silk glands. BmDuox mRNA was expressed during all the developmental stages of the silkworm. Subcellular localization revealed that BmDoux was present mainly in the periphery of the cells. Some cytoplasmic staining was detected, with rare signals in the nucleus. Expression of BmDuox was induced significantly in the larval midgut upon challenge by Escherichia coli and Bombyx mori nucleopolyhedrovirus (BmNPV). BmDuox-deleted larvae showed a marked increase in microbial proliferation in the midgut after ingestion of fluorescence-labeled bacteria compared to the control. We conclude that reducing BmDuox expression greatly increased the bacterial load, suggesting BmDuox has an important role in inhibiting microbial proliferation and the maintenance of homeostasis in the silkworm midgut.

Succinate dehydrogenase assembly factor 2 is needed for assembly and activity of mitochondrial complex II and for normal root elongation in Arabidopsis

Mitochondria complex II (succinate dehydrogenase, SDH) plays a central role in respiratory metabolism as a component of both the electron transport chain and the tricarboxylic acid cycle. We report the identification of an SDH assembly factor by analysis of T-DNA insertions in At5g51040, a protein with unknown function that was identified by mass spectrometry analysis as a low abundance mitochondrial protein. This gene is co-expressed with a number of genes encoding mitochondrial proteins, including SDH1-1, and has low partial sequence similarity to human SDHAF2, a protein required for flavin-adenine dinucleotide (FAD) insertion into SDH. In contrast to observations of other SDH deficient lines in Arabidopsis, the sdhaf2 line did not affect photosynthetic rate or stomatal conductance, but instead showed inhibition of primary root elongation with early lateral root emergence, presumably due to the low SDH activity caused by the reduced abundance of SDHAF2. Both roots and leaves showed succinate accumulation but different responses in the abundance of other organic acids and amino acids assayed. Isolated mitochondria showed lowered SDH1 protein abundance, lowered maximal SDH activity and less protein-bound flavin-adenine dinucleotide (FAD) at the molecular mass of SDH1 in the gel separation. The short root phenotype and SDH function of sdhaf2 was fully complemented by transformation with SDHAF2. Application of the SDH inhibitor, malonate, phenocopied the sdhaf2 root architecture in WT. Whole root respiratory assays showed no difference between WT and sdhaf2, but micro-respirometry of the tips of roots clearly showed low oxygen consumption in sdhaf2 which could explain a metabolic deficit responsible for root tip growth.

Flavin-induced oligomerization in Escherichia coli adaptive response protein AidB.

The process known as “adaptive response” allows Escherichia coli to respond to small doses of DNA-methylating agents by upregulating the expression of four proteins. While the role of three of these proteins in mitigating DNA damage is well understood, the function of AidB is less clear. Although AidB is a flavoprotein, no catalytic role has been established for the bound cofactor. Here we investigate the possibility that flavin plays a structural role in the assembly of the AidB tetramer. We report the generation and biophysical characterization of deflavinated AidB and of an AidB mutant that has greatly reduced affinity for flavin adenine dinucleotide (FAD). Using fluorescence quenching and analytical ultracentrifugation, we find that apo AidB has a high affinity for FAD, as indicated by an apparent dissociation constant of 402.1 ± 35.1 nM, and that binding of substoichiometric amounts of FAD triggers a transition in the AidB oligomeric state. In particular, deflavinated AidB is dimeric, whereas the addition of FAD yields a tetramer. We further investigate the dimerization and tetramerization interfaces of AidB by determining a 2.8 Å resolution crystal structure in space group P32 that contains three intact tetramers in the asymmetric unit. Taken together, our findings provide strong evidence that FAD plays a structural role in the formation of tetrameric AidB.

ENOS–ERα Complex Goes to Telomerase

See related article, pages 34–42 Nitric oxide (NO) plays an important role not only in physiological conditions,1 such as vasodilation, inhibition of platelet aggregation, and regulation of gene transcription,2 but also in atherosclerosis development. NO is synthesized from L-arginine by a family of 3 NO synthases (NOS): neuronal (nNOS),3 inducible (iNOS),4 and endothelial (eNOS).5 eNOS possesses an N-terminal oxygenase domain containing single heme and tetrahydrobioperin (BH-4)-binding sites, a C-terminal reductase domain containing single binding sites for flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and NADPH, and a central calmodulin (CaM) binding site.6 eNOS is specifically and constitutively expressed in endothelial cells normally localized in caveolae, endoplamic reticulum, and nuclear envelop.7 In caveolae, eNOS associates with growth factor or hormone receptors. On ligand-receptor binding, the receptor-associated eNOS will be phosphorylated, homodimerized, and coupled with CaM together with cofactors BH4, heme, FAD, FMN, and NADPH to form a complex. The complex will be translocated to endoplamic reticulum via caveolin and oxidize L-arginine to release NO. However, under oxidative stress conditions, caused by atherosclerotic risk factors—such as cholesterol overloading, oxidized LDL, smoking, diabetes mellitus, etc—eNOS will be uncoupled to produce superoxide.7,8 Thus, eNOS can produce both NO and superoxide, exerting atheroprotective and proatherogenic effects, by which it modulates gene transcription. Recently, several reports have shown that activated eNOS can translocate into nucleus where it regulates gene transcription.9–12 However, the underlying mechanism remains unclear. Estrogen is an important atheroprotective molecule, possessing multiple biological effects on vasculature. There are two estrogen receptor (ER) isoforms, ER alpha (ERα) and ER beta (ERβ), residing in caveolae associated with eNOS. On ligand binding, activated ERα …

Hyperactive mutants of mouse d-aspartate oxidase: mutagenesis of the active site residue serine 308

The role of Ser-308 of murine D-aspartate oxidase (mDASPO), particularly its side chain hydroxyl group, was investigated through the use of site-specific mutational analysis of Ser-308. Recombinant mDASPO carrying a substitution of Gly, Ala, or Tyr for Ser-308 was generated, and fused to either His (His-mDASPO), or glutathione S-transferase, His, and S (GHS-mDASPO) at its N-terminus. Wild-type His-mDASPO or GHS-mDASPO or their mutant derivatives were expressed in Escherichia coli and purified by affinity chromatography. All purified recombinant proteins had functional DASPO activity. The Gly-308 and Ala-308 mutants had significantly higher catalytic efficiency towards D-Asp and N-methyl-D-Asp, and a higher affinity for flavin adenine dinucleotide (FAD) compared to the wild-type enzyme. The Tyr-308 mutant had lower catalytic efficiency and binding capacity. These results suggest that the side chain hydroxyl group of a critical residue of mDASPO, Ser-308, down-regulates enzymatic activity, substrate binding, and FAD binding. This study provides information on the active site of DASPO that will considerably enhance our understanding of the biological significance of this enzyme.

L -Sorbose Reductase and Its Transcriptional Regulator Involved in l -Sorbose Utilization of Gluconobacter frateurii

Upstream of the gene for flavin adenine dinucleotide (FAD)-dependent D-sorbitol dehydrogenase (SLDH), sldSLC, a putative transcriptional regulator was found in Gluconobacter frateurii THD32 (NBRC 101656). In this study, the whole sboR gene and the adjacent gene, sboA, were cloned and analyzed. sboR mutation did not affect FAD-SLDH activity in the membrane fractions. The SboA enzyme expressed and purified from an Escherichia coli transformant showed NADPH-dependent L-sorbose reductase (NADPH-SR) activity, and the enzyme was different from the NADPH-SR previously reported for Gluconobacter suboxydans IFO 3291 in molecular size and amino acid sequence. A mutant defective in sboA showed significantly reduced growth on L-sorbose, indicating that the SboA enzyme is required for efficient growth on L-sorbose. The sboR mutant grew on L-sorbose even better than the wild-type strain did, and higher NADPH-SR activity was detected in cytoplasm fractions. Reverse transcription-PCR experiments indicated that sboRA comprises an operon. These data suggest that sboR is involved in the repression of sboA, but not in the induction of sldSLC, on D-sorbitol and that another activator is required for the induction of these genes by D-sorbitol or L-sorbose.

Mercuric reductase structural genes from plasmid R100 and transposon Tn 501: functional domains of the enzyme

The nucleotide sequence for the 2240 bp of plasmid R100 following the merC gene of the mercuric resistance operon has been determined and compared with the homologous sequence of transposon Tn507. The sequences following merC and preceding the next structural gene merA are unrelated between R100 and Tn501 and differ in length, with 72 bp in Tn 501 and 509 bp in R100. The R100 sequence has a potential open reading frame (ORF) for a 140 amino acid polypeptide with a reasonable translational start signal preceding it. The merA genes contain 1686 (Tn 5O1) and 1695 (R100) bp respectively. When optimally aligned, the merA sequences differ in 18% of their positions. These differences were clustered in specific regions. In addition, there was one nucleotide triplet in the Tn501 sequence which has no counterpart in the R100 sequence and one dodecylnucleotide sequence in the R100 sequence without counterpart in Tn 501. Thus the predicted merA polypeptide of Tn 501 contains 561 amino acids and the R100 counterpart contains 564 amino acids. Comparison of the R100 mercuric reductase sequences with that for human glutathione reductase [Krauth-Siegel et al.: Eur. J. Biochem. 121 (1982) 259–267], for which there is a 2 Å resolution electron density map [Thieme et al. : J. Mol. Biol. 152 (1981) 763–782] shows a strong homology, with 26% identical amino acids and many conservative substitutions. This homology allows the conclusion that the active site of these enzymes and the contact positions for flavin adenine dinucleotide (FAD) and NADPH are highly conserved, while the amino- and carboxyl-terminal sequences differ.

Resonance coherent anti-Stokes Raman scattering spectra of fluorescent biological chromophores: Vibrational evidence for hydrogen bonding of flavin to glucose oxidase and for rapid solvent exchange

Coherent anti-Stokes Raman scattering (CARS) spectra are reported for flavin adenine dinucleotide (FAD) and glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase; EC 1.1.3.4), in resonance with the 450 nm flavin absorption band. Several isoalloxazine ring modes were observed (1635, 1584, 1507, 1416, and 1359 cm(-1)). A 12 cm(-1) increase in one component of the 1359 cm(-1) band upon binding to glucose oxidase was attributed to hydrogen bonding of the N3 proton to a protein acceptor. This interpretation is consistent with deuteration results. Smaller decreases (5-7 cm(-1)) on binding were observed for the 1635 and 1416 cm(-1) modes (and also a 1297 cm(-1) mode of deuterated FAD) and were attributed to environmental effects. Deuteration of bound FAD was observed within 5 min of mixing glucose oxidase with D(2)O, demonstrating ready access to solvent. Lorentzian CARS peaks were observed with omega(as) at the peak of the 450 nm absorption band, a condition which corresponds to maximum resonance enhancement if the peak corresponds to the envelope of 0-1 vibronic transitions. If omega(1) was tuned to the peak, then dispersion lineshapes were observed, reflecting a loss of Raman enhancement relative to the electronic background.

The Enzyme-like Reaction Catalyzed by Flavin-Reduced Keratin Systems

Abstract The reduced keratin (RK), which was obtained by the reductive cleavage of human hair, was found to act as an apoenzyme and to furnish the reaction field for flavin adenine dinucleotide (FAD): the absorbance of the succinic acid–FAD–RK system in solution increased in the region of 200–250 nm with the lapse of time. From this system fumaric acid was isolated. FAD was found to form a complex with RK and the FAD–RK complex exhibited a catalytic activity for the dehydrogenation reaction of succinic acid to fumaric acid. FAD was reduced in this reaction and the reduced FAD was oxidized by oxygen. It was found that the process of the dehydrogenation reaction obeyed the Michaelis-Menten kinetics; the equilibrium constants of the complex formation between FAD and RK were determined by measuring the initial velocities of the dehydrogenation reaction. The binding site of FAD and RK in the complex was discussed.