Soluble Flavin Research Articles

Renalase: Gene polymorphism and its association with hypertension in some diseases

Hypertension is a significant public health problem due to its high prevalence and association with increased risk of cardiovascular disease (CVD), and thus the major cause of death in developed countries. Most of diabetes mellitus and hemodialysis patients are expected to have hypertension and of around 10% of pregnant women have hypertension, which increases preeclampsia; the most important medical problem that threatened the life of maternal and neonatal. Previous studies showed that genetic factors could play an important role in predicting hypertension. Recently, a novel soluble flavin adenine dinucleotide (FAD)-dependent amine oxidase, called renalase, found to decrease blood pressure by degrading catecholamines. It is secreted by the kidney and is found in the heart, small intestine, skeletal muscle, endothelium, and nervous system. Renalase polymorphism of the renalase gene may affect the renalase activity and increase susceptibility to some diseases. This review highlights the structure, function, polymorphisms of renalase, and its association with hypertension in hemodialysis, cardiovascular, preeclampsia, and diabetes mellitus patients. As a conclusion, the rs10887800, rs2576178, and rs2296545 renalase gene polymorphism could thus be a risk factor for hypertension.

Addition of Riboflavin-Coupled Magnetic Beads Increases Current Production in Bioelectrochemical Systems via the Increased Formation of Anode-Biofilms.

Shewanella oneidensis is one of the best-understood model organisms for extracellular electron transfer. Endogenously produced and exported flavin molecules seem to play an important role in this process and mediate the connection between respiratory enzymes on the cell surface and the insoluble substrate by acting as electron shuttle and cytochrome-bound cofactor. Consequently, the addition of riboflavin to a bioelectrochemical system (BES) containing S. oneidensis cells as biocatalyst leads to a strong current increase. Still, an external application of riboflavin to increase current production in continuously operating BESs does not seem to be applicable due to the constant washout of the soluble flavin compound. In this study, we developed a recyclable electron shuttle to overcome the limitation of mediator addition to BES. Riboflavin was coupled to magnetic beads that can easily be recycled from the medium. The effect on current production and cell distribution in a BES as well as the recovery rate and the stability of the beads was investigated. The addition of synthesized beads leads to a more than twofold higher current production, which was likely caused by increased biofilm production. Moreover, 90% of the flavin-coupled beads could be recovered from the BESs using a magnetic separator.

MUTATION STUDY OF CELLOBIOSE DEHYDROGENASE FROM PHANEROCAETE CRHYSOSPORIUM IN A PROKARIOTIC SYSTEM FOR MULTIPLE BIOSENSOR APPLICATION

The active site of cellobiose dehydrogenase from Phanerochaete chrysosporium is composed of two subsites, catalytic C and substrate-binding B subsite. The soluble flavin domain of the Phanerochaete chrysosporium Cellobiose dehydrogenase (CDH) has been mutated in residue F282 and successfully expressed in Escherichia coli. Substitution of Phe282 to Ala, Asp, and His shown to affect its enzymatic activity and altered the enzyme’s substrate specificity. While the wild-type cellobiose dehydrogenase efficiently oxidizes only cellobiose and lactose, the three mutated Phe282 shown significant activity for glucose and maltose. For all mutants type, cellobiose has retain its maximum activity but greatly decreased with lactose. The ability of CDH to recognize glucose provide great opportunities for the application in multibiosensor. Keywords : cellobiose dehidrogenase, Glukosa, Laktosa, Maltosa, Phanerochaete chrysosporium

Identification and characterization of a FAD-dependent putrescine N-hydroxylase (GorA) from Gordonia rubripertincta CWB2

A putrescine N-hydroxylase from Gordonia rubripertincta CWB2 (GorA), a microbial N-hydroxylating monooxygenase (NMO), specific for a range of diamines (putrescine>cadaverine>hexamethylenediamine) was identified. This NMO clustered together with some known but yet to be characterized diamine NMOs which are RhbE, from Sinorhizobium meliloti 1021; AlcA, from Bordetella bronchiseptica RB50, and DesB, from Streptomyces scabiei 87-22. It comprises 459 amino acids in length and has approximately a molecular weight of 51.4kDa. It has been successfully cloned, overexpressed, and purified as a soluble flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) dependent His10-tagged protein using Escherichia coli as the cloning and expression host and pET16bP as vector. The NAD(P)H oxidation assay and a hydroxylation assay were used to assess its biochemical properties. The pH optimum is between the range of 7.0–8.0 in a potassium phosphate buffer. 1,4-diaminobutane (putrescine) was the best substrate concerning GorA activity. With the NADPH oxidation assay, the kinetic parameters of this enzyme showed an apparent Km and kcat of 361.6±0.1μM and 0.266±0.011s−1, respectively, whereas the hydroxylation assay showed GorA with an apparent Km and kcat of 737.1±0.1μM and 0.210±0.001s−1. These activity data were obtained of kinetic experiments from fixing FAD and NADPH and varying the concentration of 1,4-diaminobutane. Thus this is the first diamine N-hydroxylating monooxygenase characterized with a physiological role in siderophore biosynthesis.

Effects of soluble flavin on heterogeneous electron transfer between surface-exposed bacterial cytochromes and iron oxides

Dissimilatory iron-reducing bacteria can utilize insoluble Fe(Mn)-oxides as a terminal electron acceptor under anaerobic conditions. For Shewanella species specifically, evidence suggests that iron reduction is associated with the secretion of flavin mononucleotide (FMN) and riboflavin. However, the exact mechanism of flavin involvement is unclear; while some indicate that flavins mediate electron transfer (Marsili et al., 2008), others point to flavin serving as co-factors to outer membrane proteins (Okamoto et al., 2013). In this work, we used methyl viologen (MV+)-encapsulated, porin-cytochrome complex (MtrCAB) embedded liposomes (MELs) as a synthetic model of the Shewanella outer membrane to investigate the proposed mediating behavior of microbially produced flavins. The reduction kinetics of goethite, hematite and lepidocrocite (200μM) by MELs ([MV+]∼40μM and MtrABC⩽1nM) were determined in the presence FMN at pH 7.0 in N2 atmosphere by monitoring the concentrations of MV+ and FMN through their characteristic UV–visible absorption spectra. Experiments were performed where (i) FMN and Fe(III)-oxide were mixed and then reacted with the reduced MELs and (ii) FMN was reacted with the reduced MELs followed by addition of Fe(III)-oxide. The redox reactions proceeded in two steps: a fast step that was completed in a few seconds, and a slower one lasting over 400s. For all three Fe(III)-oxides, the initial reaction rate in the presence of a low concentration of FMN (⩽1μM) was at least a factor of five faster than those with MELs alone, and orders of magnitude faster than those by FMNH2, suggesting that FMN may serve as a co-factor that enhances electron transfer from outer-membrane c-cytochromes to Fe(III)-oxides. The rate and extent of the initial reaction followed the order of lepidocrocite>hematite>goethite, the same as their reduction potentials, implying thermodynamic control on reaction rate. For LEP, with the highest reduction potential among the three Fe(III)-oxides, its reduction by FMNH2 was completed in less than 10min, suggesting that FMN was capable of mediating electron transfer to LEP. At higher FMN concentrations (>1μM), the reaction rates for both steps decreased and varied inversely with FMN concentration, indicating that FMN inhibited the MEL to Fe(III)-oxide electron transfer reaction under these conditions. The implications of the observed kinetic behaviors to flavin-mediated Fe(III)-oxide reduction in natural environments are discussed.

In silico Analyses of Flavin Reductase from Citrobacter freundii A1

Objective: The aim of the present study was to describe the structural and phylogenetic features of flavin reductase from C.freundii Al using bioinfonnatics tools. Materials and methods:The flavin reductase (Ire) gene from a dye-degrading bacterium, C. freundii Al was isolated and amplified by Polymerase Chain Reaction (peR). The gene encodes for NAD(P)H:flavin oxidoreductase, an enzyme that catalyzes the reduction of soluble flavins by reduced pyridine nucleotides this was believed to be the azoreductase from C. freundii AI. The gene, approximately 0.8 kb was sequenced and in silica analyses of the nucleotide sequence were performed. Results: From phlyogenetic analyses, we observed that flavin reductase enzyme generally existed in most microorganisms and the enzyme from C.freundii Al is conserved among the Gram-negative bacteria. The protein function of the flavin reductase coded was predicted based on the motifs of deduced amino acid sequence. The amino acid sequence of flavin reductase from C. freundii Al was compared with other azoreductases and was found to be a unique NADPH-preferred azoreductase. Conclusion: Hence, in silica characterization of flavin reductase gene presented various features of the gene and this would facilitate molecular studies of the gene and reveal the functional role of the enzyme.

Functional expression of Phanerochaete chrysosporium cellobiose dehydrogenase flavin domain in Escherichia coli

Cellobiose dehydrogenase (CDH; EC 1.1.99.18) is an extracellular glycosylated protein composed of two distinct domains, a C-terminal catalytic flavin domain and an N-terminal cytochrome-b-type heme domain, which transfers electrons from the flavin domain to external electron acceptors. The soluble flavin domain of the Phanerochaete chrysosporium CDH was successfully expressed in Escherichia coli. The enzyme showed dye-mediated CDH activity higher than that of the complete CDH, composed of flavin domain and heme domain, prepared using Pichia pastoris as the host microorganism. The ability to conveniently express the recombinant CDH flavin domain in E. coli provides great opportunities for the molecular engineering of the catalytic properties of CDH.

Electrochemical Measurement of Electron Transfer Kinetics by Shewanella oneidensis MR-1

Shewanella oneidensis strain MR-1 can respire using carbon electrodes and metal oxyhydroxides as electron acceptors, requiring mechanisms for transferring electrons from the cell interior to surfaces located beyond the cell. Although purified outer membrane cytochromes will reduce both electrodes and metals, S. oneidensis also secretes flavins, which accelerate electron transfer to metals and electrodes. We developed techniques for detecting direct electron transfer by intact cells, using turnover and single turnover voltammetry. Metabolically active cells attached to graphite electrodes produced thin (submonolayer) films that demonstrated both catalytic and reversible electron transfer in the presence and absence of flavins. In the absence of soluble flavins, electron transfer occurred in a broad potential window centered at approximately 0 V (versus standard hydrogen electrode), and was altered in single (DeltaomcA, DeltamtrC) and double deletion (DeltaomcA/DeltamtrC) mutants of outer membrane cytochromes. The addition of soluble flavins at physiological concentrations significantly accelerated electron transfer and allowed catalytic electron transfer to occur at lower applied potentials (-0.2 V). Scan rate analysis indicated that rate constants for direct electron transfer were slower than those reported for pure cytochromes (approximately 1 s(-1)). These observations indicated that anodic current in the higher (>0 V) window is due to activation of a direct transfer mechanism, whereas electron transfer at lower potentials is enabled by flavins. The electrochemical dissection of these activities in living cells into two systems with characteristic midpoint potentials and kinetic behaviors explains prior observations and demonstrates the complementary nature of S. oneidensis electron transfer strategies.

Riboflavin Is an Active Redox Cofactor in the Na+-pumping NADH:Quinone Oxidoreductase (Na+-NQR) from Vibrio cholerae

Here we present new evidence that riboflavin is present as one of four flavins in Na+-NQR. In particular, we present conclusive evidence that the source of the neutral radical is not one of the FMNs and that riboflavin is the center that gives rise to the neutral flavosemiquinone. The riboflavin is a bona fide redox cofactor and is likely to be the last redox carrier of the enzyme, from which electrons are donated to quinone. We have constructed a double mutant that lacks both covalently bound FMN cofactors (NqrB-T236Y/NqrC-T225Y) and have studied this mutant together with the two single mutants (NqrB-T236Y and NqrC-T225Y) and a mutant that lacks the noncovalently bound FAD in NqrF (NqrF-S246A). The double mutant contains riboflavin and FAD in a 0.6:1 ratio, as the only flavins in the enzyme; noncovalently bound flavins were detected. In the oxidized form, the double mutant exhibits an EPR signal consistent with a neutral flavosemiquinone radical, which is abolished on reduction of the enzyme. The same radical can be observed in the FAD deletion mutant. Furthermore, when the oxidized enzyme reacts with ubiquinol (the reduced form of the usual electron acceptor) in a process that reverses the physiological direction of the electron flow, a single kinetic phase is observed. The kinetic difference spectrum of this process is consistent with one-electron reduction of a neutral flavosemiquinone. The presence of riboflavin in the role of a redox cofactor is thus far unique to Na+-NQR.

Characterization of a Novel Bifunctional Dihydropteroate Synthase/Dihydropteroate Reductase Enzyme from Helicobacter pylori

Tetrahydrofolate is a ubiquitous C(1) carrier in many biosynthetic pathways in bacteria, importantly, in the biosynthesis of formylmethionyl tRNA(fMet), which is essential for the initiation of translation. The final step in the biosynthesis of tetrahydrofolate is carried out by the enzyme dihydrofolate reductase (DHFR). A search of the complete genome sequence of Helicobacter pylori failed to reveal any sequence that encodes DHFR. Previous studies demonstrated that the H. pylori dihydropteroate synthase gene folP can complement an Escherichia coli strain in which folA and folM, encoding two distinct DHFRs, are deleted. It was also shown that H. pylori FolP possesses an additional N-terminal domain that binds flavin mononucleotide (FMN). Homologous domains are found in FolP proteins of other microorganisms that do not possess DHFR. In this study, we demonstrated that H. pylori FolP is also a dihydropteroate reductase that derives its reducing power from soluble flavins, reduced FMN and reduced flavin adenine dinucleotide. We also determined the stoichiometry of the enzyme-bound flavin and showed that half of the bound flavin is exchangeable with the soluble flavins. Finally, site-directed mutagenesis of the most conserved amino acid residues in the N-terminal domain indicated the importance of these residues for the activity of the enzyme as a dihydropteroate reductase.

Chemistry for an essential biological process: the reduction of ferric iron.

In biological systems, the predominant form of iron is the trivalent Fe(III) form, which is potentially not readily bioavailable because of its hydrolysis and polymerization to insoluble forms. It is also the easiest of the two predominant forms of iron to chelate selectively. In a short overview of iron chemistry, we point out some of the pitfalls using standard redox potentials, comment on the interaction of ferric complexes with hydrogen peroxide to give hydroxyl radicals and address the release of iron from ferrisiderophores. In biological systems there are two classes of ferric reductases, the soluble flavin reductases found in prokaryotes, and the membrane-bound cytochrome b-like reductases found in eukaryotes. Finally the role of dissimilatory ferric reduction in microbial respiration and biomineralization is discussed.

Characterization of the flavin reductase gene (fre) of Escherichia coli and construction of a plasmid for overproduction of the enzyme

The enzyme NAD(P)H:flavin oxidoreductase (flavin reductase) catalyzes the reduction of soluble flavins by reduced pyridine nucleotides. In Escherichia coli it is part of a multienzyme system that reduces the Fe(III) center of ribonucleotide reductase to Fe(II) and thereby sets the stage for the generation by dioxygen of a free tyrosyl radical required for enzyme activity. Similar enzymes are known in other organisms and may more generally be involved in iron metabolism. We have now isolated the gene for the E. coli flavin reductase from a lambda gt11 library. After DNA sequencing we found an open reading frame coding for a polypeptide of 233 amino acids, with a molecular weight of 26,212 and with an N-terminal segment identical to that determined by direct Edman degradation. The coding sequence is preceded by a weak ribosome binding site centered 8 nucleotides from the start codon and by a promoterlike sequence centered at a distance of 83 nucleotides. In a Kohara library the gene hybridized to position 3680 on the physical map of E. coli. A bacterial strain that overproduced the enzyme approximately 100-fold was constructed. The translated amino acid sequence contained a potential pyridine nucleotide-binding site and showed 25% identity with the C-terminal part of one subunit (protein C) of methane monooxygenase from methanotropic bacteria that reduces the iron center of a second subunit (protein A) of the oxygenase by pyridine nucleotides.

Acyl Transfer from S-Monoacyldihydrolipoamide to Benzylamine in the Presence of Oxidizing Agents

Abstract Acyl transfer reaction from N-benzyl-8-S-(p-nitrobenzoyl)dihydrolipoamide to benzylamine was enhanced with oxidizing agents such as cobalt(II) ion or a soluble flavin under an oxygen atmosphere or with di-4-pyridyl disulfide, accompanied by the formation of N-benzyllipoamide.

In-vitro riboflavin binding and endogenous flavins in Phycomyces blakesleeanus

Several types of membrane-localized flavin binding sites were investigated in sporangiophores (spph) and mycelia of Phycomyces blakesleeanus. In-vitro binding of riboflavin, riboflavin-5'-phosphate, and flavin-adenine-dinucleotide was demonstrated with unfractionated membrane preparations by means of competition of [(14)C]riboflavin binding. Saturation of binding was only obtained with the highly water-soluble riboflavin-5'-phosphate, but by extrapolation it was shown that riboflavin showed the highest affinity towards the binding sites (KD about 4·10(-6)M). The number of binding sites was estimated to be 0.7 nmol g(-1) fresh-weight equivalent. Analysis of endogenous soluble flavin revealed that only riboflavin, riboflavin-5'-phosphate, and flavin-adenine-dinucleotide occurred in Phycomyces, and at a concentration of at least 1 nmol g(-1) fresh-weight equivalent in entire spph. Thus, the measured binding sites could reach saturation in-vivo. In the apical part of spph to which blue-light sensitivity is restricted, the amount of soluble flavin was three-fold higher. Exclusively in this zone, heat-labile riboflavin proteins were measured at a concentration of about 3 nmol g(-1) fresh-weight equivalent. The amount of covalently bound flavin was higher in spph tips than in intact spph (8 nmol and 3 nmol g(-1) fresh-weight equivalent, respectively). In either case, the concentrations of the flavin-membrane complexes were higher than the theoretical calculated concentration of (anisotropic) blue-light photoreceptor in Phycomyces (Bergman et al. 1969), and their involvement in blue-light photoreception is considered.

A continuous spectrophotometric determination of hepatic microsomal azo reductase activity and its dependence on cytochrome P-450.

1. A continuous spectrophotometric determination of rat hepatic microsomal anaerobic azo reductase activity has been developed. 2. The addition of soluble flavins (riboflavin, FMN or FAD) greatly increased this NADPH-dependent activity towards a number of azo substrates. 3. Investigations with amaranth as substrate gave an apparent Km of 34 microM and Vmax. of 4 nmol/min per mg of microsomal protein. The inclusion of a fixed concentration of FMN increased Vmax. and greatly decreased Km, the magnitude of these changes reflecting the concentration of flavin present. 4. Investigations using a fixed amaranth concentration over a range of flavin concentrations gave biphasic double-reciprocal plots with two apparent Km and Vmax. values. 5. Pretreatment of animals with cobaltous chloride, 2-allyl-2-isopropylacetamide, carbon tetrachloride, phenobarbitone and 3-methylcholanthrene altered azo reductase activity in parallel with changes in cytochrome P-450 content. 6. The significance of these results is discussed in terms of the electron-transfer components present in the hepatic microsomal fraction.

Membrane-bound respiratory chain of Pseudomonas aeruginosa grown aerobically.

The electron transport chain of the gram-negative bacterium Pseudomonas aeruginosa, grown aerobically, contained a number of primary dehydrogenases and respiratory components (soluble flavin, bound flavin, coenzyme Q9, heme b, heme c, and cytochrome o) in membrane particles of the organism. Cytochrome o, about 50% of the b-type cytochrome, seemed to function as a terminal oxidase in the respiratory chain. The electron transport chain of P. aeruginosa grown aerobically was suggested to be lined up in order of primary dehydrogenase, b, c1, c, o, and oxygen.

In vitro binding of riboflavin to subcellular particles from maize coleoptiles and Cucurbita hypocotyls

Saturable and reversible in vitro binding of [(14)C]riboflavin was found to occur on subcellular, sedimentable particles from maize coleoptiles and Cucurbita hypocotyls. The KD was ca. 6 μM, the pH optimum was near 6.0, and the number of binding sites amounted to 0.1-0.5 μM on a fresh-weight basis. When the reducing agent dithionite was present, riboflavin binding increased-the KD was 2.5 μM, and the pH optimum above 8.0. The binding was specific: flavin mononucleotide (FMN) and flavin adenosine-dinucleotide (FAD) bound less tightly to these sites than riboflavin and another major soluble flavin, the previously described riboflavin-analog "FX", occurring in grass coleoptiles. These flavin-binding sites were localized on vesicles derived from plasmalemma and endoplasmic reticulum by analyzing sucrose and metrizamide density gradients and marker enzymes.

METABOLIC POOLS IN THE SPADIX OF ARUM DURING FLORAL DEVELOPMENT

SUMMARYThe concentration of certain metabolic intermediates in the spadix of Arum has been determined over the complete period of floral development from the point at which the spathes are first visible in the leaf base to thermogenesis in the spadix. These intermediates include sucrose, glucose, fructose, pyruvate, alanine, citrate, α‐ketoglutarate, glutamate, glutamine, malate, aspartate and asparagine. Assays for these intermediates were supplemented by measurements of mitochondrial respiratory activity, soluble and protein‐bound flavins, thiamine and ethanol‐insoluble materials.These constituents and processes reveal four broad patterns of change indicating a complex system controlling floral development and thermogenesis. The clearest indication of a specific control seen is the correlation of low mitochondrial respiratory activity with low levels of soluble flavins, suggesting that lack of these cofactors limits the respiratory activity of young spadices.

Mechanisms of azo reduction by Streptococcus faecalis. II. The role of soluble flavins.

1. Using a previously developed continuous assay system, the mechanism of stimulation of Red 2G azo reduction in Streptococcus faecalis by additions of soluble flavins has been investigated.2. Reduced flavins acting as two-electron donors can rapidly reduce Red 2G non-enzymically and the reduced flavins can act as an electron shuttle from NAD(P)H-dependent flavoproteins to the acceptor azo compound.3. Results of inhibition studies were consistent with the direct participation of soluble flavins and the non-involvement of cytochromes in azo reduction.4. Enzyme fractions possessing azo-reductase activity also showed cytochrome c reductase and diaphorase activity, and respired oxygen. Azo reduction under anaerobic conditions may represent a fortuitous, non-enzymic reduction by enzymically-generated reduced flavins; inhibition by oxygen being due to regeneration of the oxidized form.

Enzymes catalyzing sequential reactions in mouse brain and liver supernatant fractions: I. Differential use of Janus green B and phenazine methosulfate.

(1) Janus green B and phenazine methosulfate were used to determine the presence of single enzymes, or enzyme systems, which catalyze sequential reactions in brain and liver supernatants. (2) Spectrophotometric studies on the reduction of Janus green B by supernatants demonstrated the absence of functional flavoprotein in brain supernatants and their presence in liver supernatants, corroborating results obtained by manometry. (3) Brain supernatants contained at least 0.5 microgram of total flavin per ml equivalent; liver supernatant contained about 7 micrograms per ml equivalent. Therefore, the inability of brain supernatant to react with Janus green B was due not to the absence of riboflavin or flavin-adenine dinucleotide, but to the absence of a functional flavoprotein. (4) The absence of a functional flavoprotein did not eliminate the activity of the glycolytic enzyme system in brain supernatants as measured by substrate-dependent lactate accumulation. (5) A flavin inhibitor, acriflavin, did not significantly block glycolysis in brain or liver supernatants, either in the presence or absence of Janus green B. (6) There was no evidence for the presence of a soluble flavin inhibitor in brain supernatants. (7) Reduced diphosphopyridine nucleotide, used as substrate, was oxidized endogenously at a slow rate by both supernatants. This rate was increased by Janus green B in liver but not in brain supernatants. (8) In the presence of pyruvate and Janus green B, both supernatants rapidly oxidized reduced diphosphopyridine nucleotide, but the dye remained oxidized. This indicates that the flavindye-oxygen electron transport chain was inoperative in the presence of the lactic dehydrogenase-pyruvate system of both supernatants. (9) Where the absence of functional flavoprotein prevented the use of Janus green B for the demonstration of enzyme systems, phenazine methosulfate was used. Oxygen uptake in the presence of this dye was a measure of reduced diphosphopyridine nucleotide formed. Thus, it was a measure of the activity of enzymes or enzyme systems, of which diphosphopyridine nucleotide-requiring dehydrogenases are components. (10) By supplying phenazine methosulfate as terminal electron transport agent, both supernatants were shown to contain enzyme systems which metabolized fructose diphosphate, lactate, and malate. (11) The results are discussed with reference to the thesis that the enzyme systems occurring in supernatants represent fractions of those retained by the corresponding mitochondria.