Flavin Mononucleotide Reductase Research Articles

Cloning of Nitrate Reductase and Nitrite Reductase Genes and Their Functional Analysis in Regulating Cr(VI) Reduction in Ectomycorrhizal Fungus Pisolithus sp.1.

Assimilatory-type nitrate reductase (NR) and nitrite reductase (NiR) are the key enzymes that involve in nitrate assimilation and nitrogen cycling in microorganisms. NR and NiR with NADH or NADPH and FMN or FAD domains could be coupled to the reduction process of hexavalent chromium [Cr(VI)] in microorganisms. A new assimilatory-type NR gene (named niaD) and a new assimilatory-type NiR gene (named niiA) are cloned, identified, and functionally characterized by 5′ and 3′ RACE, alignment, annotation, phylogenetic tree, and yeast mutant complementation analyses from Pisolithus sp.1, a dominant symbiotic ectomycorrhizal fungi (EMF) that can assist in phytoremediation. Assimilatory-type niaD and niiA were 2,754 bp and 3,468 bp and encode a polypeptide with 917 and 1,155 amino acid residues, respectively. The isoelectric points of NR (Pisolithus sp.1 NR) and NiR (Pisolithus sp.1 NiR) of Pisolithus sp.1 are 6.07 and 6.38, respectively. The calculated molecular mass of Pisolithus sp.1 NR and Pisolithus sp.1 NiR is 102.065 and 126.914 kDa, respectively. Yeast mutant complementation analysis, protein purification, and activities of NR and NiR under Cr treatment suggest that Pisolithus sp.1 NR is a functional NR that mediates Cr(VI) tolerance and reduction. The multiple alignment demonstrates that Pisolithus sp.1 NR is potentially a nicotinamide adenine dinucleotide phosphate-dependent flavin mononucleotide reductase and also Class II chromate reductase. Our results suggest that Pisolithus sp.1 NR plays a key role in Cr(VI) reduction in the EMF Pisolithus sp.1.

NfoR: Chromate Reductase or Flavin Mononucleotide Reductase?

Soil bacteria can detoxify Cr(VI) ions by reduction. Within the last 2 decades, numerous reports of chromate reductase enzymes have been published. These reports describe catalytic reduction of chromate ions by specific enzymes. These enzymes each have sequence similarity to known redox-active flavoproteins. We investigated the enzyme NfoR from Staphylococcus aureus, which was reported to be upregulated in chromate-rich soils and to have chromate reductase activity (H. Han, Z. Ling, T. Zhou, R. Xu, et al., Sci Rep 7:15481, 2017, https://doi.org/10.1038/s41598-017-15588-y). We show that NfoR has structural similarity to known flavin mononucleotide (FMN) reductases and reduces FMN as a substrate. NfoR binds FMN with a dissociation constant of 0.4 μM. The enzyme then binds NADPH with a dissociation constant of 140 μM and reduces the flavin at a rate of 1,350 s-1 Turnover of the enzyme is apparently limited by the rate of product release that occurs, with a net rate constant of 0.45 s-1 The rate of product release limits the rate of observed chromate reduction, so the net rate of chromate reduction by NfoR is orders of magnitude lower than when this process occurs in solution. We propose that NfoR is an FMN reductase and that the criterion required to define chromate reduction as enzymatic has not been met. That NfoR expression is increased in the presence of chromate suggests that the survival adaption was to increase the net rate of chromate reduction by facile, adventitious redox processes.IMPORTANCE Chromate is a toxic by-product of multiple industrial processes. Chromate reduction is an important biological activity that ameliorates Cr(VI) toxicity. Numerous researchers have identified chromate reductase activity by observing chromate reduction. However, all identified chromate reductase enzymes have flavin as a cofactor or use a flavin as a substrate. We show here that NfoR, an enzyme claimed to be a chromate reductase, is in fact an FMN reductase. In addition, we show that reduction of a flavin is a viable way to transfer electrons to chromate but that it is unlikely to be the native function of enzymes. We propose that upregulation of a redox-active flavoprotein is a viable means to detoxify chromate that relies on adventitious reduction that is not catalyzed.

ArsH protects Pseudomonas putida from oxidative damage caused by exposure to arsenic.

The two As resistance arsRBC operons of Pseudomonas putida KT2440 are followed by a downstream gene called arsH that encodes an NADPH-dependent flavin mononucleotide reductase. In this work, we show that the arsH1 and (to a lesser extent) arsH2 genes of P. putida KT2440 strengthened its tolerance to both inorganic As(V) and As(III) and relieved the oxidative stress undergone by cells exposed to either oxyanion. Furthermore, overexpression of arsH1 and arsH2 endowed P. putida with a high tolerance to the oxidative stress caused by diamide (a drainer of metabolic NADPH) in the absence of any arsenic. To examine whether the activity of ArsH was linked to a direct action on the arsenic compounds tested, arsH1 and arsH2 genes were expressed in Escherichia coli, which has an endogenous arsRBC operon but lacks an arsH ortholog. The resulting clones both deployed a lower production of reactive oxygen species (ROS) when exposed to As salts and had a superior endurance to physiological redox insults. These results suggest that besides the claimed direct action on organoarsenicals, ArsH contributes to relieve toxicity of As species by mediating reduction of ROS produced in vivo upon exposure to the oxyanion, e.g. by generating FMNH2 to fuel ROS-quenching activities.

440 - Electron Transfer Activity of Mitochondrial Outer Membrane Protein MitoNEET

Mitochondrial outer membrane protein MitoNEET, a primary target of type II diabetes drug pioglitazone, is a key regulator of energy metabolism in mitochondria. The protein exists as a homodimer with each monomer containing a redox active [2Fe-2S] cluster. Here we report that mitoNEET has a specific interaction with flavin mononucleotide (FMN). In the presence of flavin reductase, FMNH2 reduced by NADH can rapidly reduce the mitoNEET [2Fe-2S] clusters under anaerobic conditions. Under aerobic conditions, however, the reduced mitoNEET [2Fe-2S] clusters are constantly re-oxidized by oxygen with concomitant oxidation of NADH in the presence of FMN and flavin reductase. The reduced mitoNEET [2Fe-2S] clusters can also reduce ubiquinone-2, a ubiquinone-10 analog, under aerobic or anaerobic conditions, indicating that mitoNEET has the ability to transfer electron from NADH to the membrane-bound ubiquinone in mitochondria. The [2Fe-2S] cluster is required for the electron transfer activity of mitoNEET, as apo-form mitoNEET has no activity to mediate oxidation of NADH or reduction of ubiquinone. The pioglitazone analog NL-1, which has a specific binding affinity for mitoNEET, effectively inhibits the electron transfer activity of the mitoNEET [2Fe-2S] clusters. The results suggest that mitoNEET is a novel electron transfer protein that may directly regulate energy metabolism in mitochondria.

The reduced flavin-dependent monooxygenase SfnG converts dimethylsulfone to methanesulfinate

The biochemical pathway through which sulfur may be assimilated from dimethylsulfide (DMS) is proposed to proceed via oxidation of DMS to dimethylsulfoxide (DMSO) and subsequent conversion of DMSO to dimethylsulfone (DMSO2). Analogous chemical oxidation processes involving biogenic DMS in the atmosphere result in the deposition of DMSO2 into the terrestrial environment. Elucidating the enzymatic pathways that involve DMSO2 contribute to our understanding of the global sulfur cycle. Dimethylsulfone monooxygenase SfnG and flavin mononucleotide (FMN) reductase MsuE from the genome of the aerobic soil bacterium Pseudomonas fluorescens Pf0-1 were produced in Escherichia coli, purified, and biochemically characterized. The enzyme MsuE functions as a reduced nicotinamide adenine dinucleotide (NADH)-dependent FMN reductase with apparent steady state kinetic parameters of Km = 69 μM and kcat/Km = 9 min−1 μM −1 using NADH as the variable substrate, and Km = 8 μM and kcat/Km = 105 min−1 μM −1 using FMN as the variable substrate. The enzyme SfnG functions as a flavoprotein monooxygenase and converts DMSO2 to methanesulfinate in the presence of FMN, NADH, and MsuE, as evidenced by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. The results suggest that methanesulfinate is a biochemical intermediate in sulfur assimilation.

Cloning and sequence analysis demonstrate the chromate reduction ability of a novel chromate reductase gene from Serratia sp.

The ChrT gene encodes a chromate reductase enzyme which catalyzes the reduction of Cr(VI). The chromate reductase is also known as flavin mononucleotide (FMN) reductase (FMN_red). The aim of the present study was to clone the full-length ChrT DNA from Serratia sp. CQMUS2 and analyze the deduced amino acid sequence and three-dimensional structure. The putative ChrT gene fragment of Serratia sp. CQMUS2 was isolated by polymerase chain reaction (PCR), according to the known FMN_red gene sequence from Serratia sp. AS13. The flanking sequences of the ChrT gene were obtained by high efficiency TAIL-PCR, while the full-length gene of ChrT was cloned in Escherichia coli for subsequent sequencing. The nucleotide sequence of ChrT was submitted onto GenBank under the accession number, KF211434. Sequence analysis of the gene and amino acids was conducted using the Basic Local Alignment Search Tool, and open reading frame (ORF) analysis was performed using ORF Finder software. The ChrT gene was found to be an ORF of 567 bp that encodes a 188-amino acid enzyme with a calculated molecular weight of 20.4 kDa. In addition, the ChrT protein was hypothesized to be an NADPH-dependent FMN_red and a member of the flavodoxin-2 superfamily. The amino acid sequence of ChrT showed high sequence similarity to the FMN reductase genes of Klebsiella pneumonia and Raoultella ornithinolytica, which belong to the flavodoxin-2 superfamily. Furthermore, ChrT was shown to have a 85.6% similarity to the three-dimensional structure of Escherichia coli ChrR, sharing four common enzyme active sites for chromate reduction. Therefore, ChrT gene cloning and protein structure determination demonstrated the ability of the gene for chromate reduction. The results of the present study provide a basis for further studies on ChrT gene expression and protein function.

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 Å and 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.

Azoreductase from Rhodobacter sphaeroides AS1.1737 is a flavodoxin that also functions as nitroreductase and flavin mononucleotide reductase

Previously reported azoreductase (AZR) from Rhodobacter sphaeroides AS1.1737 was shown to be a flavodoxin possessing nitroreductase and flavin mononucleotide (FMN) reductase activities. The structure model of AZR constructed with SWISS-MODEL displayed a flavodoxin-like fold with a three-layer alpha/beta/alpha structure. With nitrofurazone as substrate, the optimal pH value and temperature were 7.0 and 50 degrees C, respectively. AZR could reduce a number of nitroaromatic compounds including 2,4-dinitrotoluene, 2,6-dinitrotoluene, 3,5-dinitroaniline, and 2,4,6-trinitrotoluene (TNT). TNT resulted to be the most efficient nitro substrate and was reduced to hydroxylamino-dinitrotoluene. Both NADH and NADPH could serve as electron donors of AZR, where the latter was preferred. Externally added FMN was also reduced by AZR via ping-pong mechanism and was a competitive inhibitor of NADPH, methyl red, and nitrofurazone. AZR with broad substrate specificity is a member of a new nitro/FMN reductase family demonstrating potential application in bioremediation.

Purification and characterization of EDTA monooxygenase from the EDTA-degrading bacterium BNC1.

The synthetic chelating agent EDTA can mobilize radionuclides and heavy metals in the environment. Biodegradation of EDTA should reduce this mobilization. Although several bacteria have been reported to mineralize EDTA, little is known about the biochemistry of EDTA degradation. Understanding the biochemistry will facilitate the removal of EDTA from the environment. EDTA-degrading activities were detected in cell extracts of bacterium BNC1 when flavin mononucleotide (FMN), NADH, and O2 were present. The degradative enzyme system was separated into two different enzymes, EDTA monooxygenase and an FMN reductase. EDTA monooxygenase oxidized EDTA to glyoxylate and ethylenediaminetriacetate (ED3A), with the coconsumption of FMNH2 and O2. The FMN reductase provided EDTA monooxygenase with FMNH2 by reducing FMN with NADH. The FMN reductase was successfully substituted in the assay mixture by other FMN reductases. EDTA monooxygenase was purified to greater than 95% homogeneity and had a single polypeptide with a molecular weight of 45,000. The enzyme oxidized both EDTA complexed with various metal ions and uncomplexed EDTA. The optimal conditions for activity were pH 7.8 and 35 degreesC. Kms were 34.1 microM for uncomplexed EDTA and 8.5 microM for MgEDTA2-; this difference in Km indicates that the enzyme has greater affinity for MgEDTA2-. The enzyme also catalyzed the release of glyoxylate from nitrilotriacetate and diethylenetriaminepentaacetate. EDTA monooxygenase belongs to a small group of FMNH2-utilizing monooxygenases that attack carbon-nitrogen, carbon-sulfur, and carbon-carbon double bonds.

Purification and Characterization of Isobutylamine N-Hydroxylase from the Valanimycin Producer Streptomyces viridifaciensMG456-hF10

Streptomyces viridifaciensMG456-hF10 produces the antitumor agent valanimycin, which is a member of a family of antibiotics containing the azoxy group. An enzyme involved in the biosynthesis of valanimycin has been purified 360-fold from S. viridifaciens.This enzyme, isobutylamine N-hydroxylase, catalyzes the oxidation of isobutylamine to isobutylhydroxylamine in the presence of oxygen and a reduced flavin cofactor. Unlike other known N-hydroxylases, isobutylamine N-hydroxylase cannot carry out the reduction of the flavin cofactor. Rather, the reduced flavin is supplied by a separate flavin reductase that is present in extracts of S. viridifaciens.The reduced flavin cofactor could also be supplied by the flavin mononucleotide reductase of Vibrio fischeri.The requirement for molecular oxygen and a reduced flavin indicates that the N-hydroxylase is a flavin monooxygenase and that the mechanism for the hydroxylation is likely to proceed via the formation of a flavin 4a-hydroperoxide. Isobutylamine N-hydroxylase exhibited a subunit molecular mass of 40 kDa and existed in dimeric or trimeric form depending upon buffer conditions. The p Iof the protein was found to be ca. 5.1 and the enzyme exhibited a sensitivity to thiol-directed reagents.

Purification and Characterization of Chorismate Synthase from Euglena gracilis

Chorismate synthase was purified 1200-fold from Euglena gracilis. The molecular mass of the native enzyme is in the range of 110 to 138 kilodaltons as judged by gel filtration. The molecular mass of the subunit was determined to be 41.7 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified chorismate synthase is associated with an NADPH-dependent flavin mononucleotide reductase that provides in vivo the reduced flavin necessary for catalytic activity. In vitro, flavin reduction can be mediated by either dithionite or light. The enzyme obtained from E. gracilis was compared with chorismate synthases purified from a higher plant (Corydalis sempervirens), a bacterium (Escherichia coli), and a fungus (Neurospora crassa). These four chorismate synthases were found to be very similar in terms of cofactor specificity, kinetic properties, isoelectric points, and pH optima. All four enzymes react with polyclonal antisera directed against chorismate synthases from C. sempervirens and E. coli. The closely associated flavin mononucleotide reductase that is present in chorismate synthase preparations from E. gracilis and N. crassa is the main difference between those synthases and the monofunctional enzymes from C. sempervirens and E. coli.

Transient kinetics of reduction of blue copper proteins by free flavin and flavodoxin semiquinones.

Rate constants have been determined for the electron-transfer reactions between reduced free flavins and flavodoxin semiquinone and several blue copper proteins. Correlations between these values and redox potentials demonstrate that spinach plastocyanin, Pseudomonas aeruginosa azurin, Alcaligenes sp. azurin, and Alcaligenes sp. nitrite reductase have the same intrinsic reactivities toward free flavins, whereas stellacyanin is more reactive (3.3 times) and laccase considerably less reactive (approximately 12 times). Electrostatic interactions between the negatively charged flavin mononucleotide (FMN) and the copper proteins show that the interaction site charges for laccase and nitrite reductase are opposite in sign to the net protein charge and that the signs and magnitudes of the charges are consistent with the known three-dimensional structures for plastocyanin and the azurins and with amino acid sequence homologies for stellacyanin. The results demonstrate that the apparent interaction site charge with flavodoxin is larger than that with FMN for plastocyanin, nitrite reductase, and stellacyanin but smaller for Pseudomonas azurin. This is interpreted in terms of a larger interaction domain for the flavodoxin reaction, which allows charged groups more distant from the actual electron-transfer site to become involved. The intrinsic reactivities of plastocyanin and azurin toward flavodoxin are the same, as was the case with FMN, but both stellacyanin and nitrite reductase are considerably less reactive than expected (approximately 2 orders of magnitude). This result suggests the involvement of steric factors with these latter two proteins which discriminate against large reactants such as flavodoxin.

Flavin mononucleotide reductase of luminous bacteria.

NAD(P)H: FMN oxidoreductase (flavin reductase) couples in vitro to bacterial luciferase. This reductase, which is also postulated to supply reduced flavin mononucleotide in vivo as a substrate for the bioluminescent reaction, has been partially purified and characterized from two species of luminous bacterial. From Photobacterium fischeri the enzyme has a M. W. determined by Sephadex gel filtration, of 43,000 and may have a subunit structure. The turnover number at 20 degrees C, based on a purity estimate of 20 percent, is 1.7 times 10-4 moles of NADH oxidized per min per mole of reductase. The reductase isolated from Beneckea harveyi has an apparent molecular weight of 23,000; its purity was too low to permit estimation of specific activity. Using a spectrophotometric assay at 340 nm with the P. fischeri reductase, both NADH (Km, 8 times 10-5 M) and NADPH (Km, 4 times 10-4 M) were enzymatically oxidized, the Vmax with NADH being approximately twice that of NADPH. Of the flavins tested in this assay, only FMN (Km, 7.3 times 10-5 M) and FAD (Km, 1.4 times 10-4 M) were effective, FMN having a Vmax three times that of FAD. In the coupled assay, i.e., measuring the bioluminescence intensity of the reaction with added luciferase, the optimum FMN concentration was nearly 100 times less than in the spectrophotometric assay. The studies reported suggest the existence of a functional reductase-luciferase complex.