Crystal structure of an aerobic FMN-dependent azoreductase (AzoA) from Enterococcus faecalis

The initial critical step of reduction of the azo bond during the metabolism of azo dyes is catalyzed by a group of NAD(P)H dependant enzymes called azoreductases. Although several azoreductases have been identified from microorganisms and partially characterized, very little is known about the structural basis for substrate specificity and the nature of catalysis. Enterococcus faecalis azoreductase A (AzoA) is a highly active azoreductase with a broad spectrum of substrate specificity and is capable of degrading a wide variety of azo dyes. Here, we report the crystal structure of the AzoA from E. faecalis determined at 2.07 A resolution with bound FMN ligand. Phases were obtained by single wavelength anomalous scattering of selenomethionine labeled protein crystals. The asymmetric unit consisted of two dimers with one FMN molecule bound to each monomer. The AzoA monomer takes a typical NAD(P)-binding Rossmann fold with a highly conserved FMN binding pocket. A salt bridge between Arg18 and Asp184 restricts the size of the flavin binding pocket such that only FMN can bind. A putative NADH binding site could be identified and a plausible mechanism for substrate reduction is proposed. Expression studies revealed azoA gene to be expressed constitutively in E. faecalis.     

Probing the NADH- and Methyl Red-binding site of a FMN-dependent azoreductase (AzoA) from Enterococcus faecalis

AzoA from Enterococcus faecalis is a member of the polymeric flavin-dependent NADH-preferred azoreductase group. Little is known about the binding and interaction of NADH and azo dye in the azoreductase group. A synergetic strategy based on computational prediction, reverse genetics validation coupled with site-directed mutagenesis, and reconstruction of mutation network was used to investigate the binding and interaction of NADH and a model azo dye, Methyl Red, with AzoA. Methyl Red and NADH interacted in a unique binding mode in which the benzoic acid moiety of Methyl Red and the nicotinamide ring of NADH were not parallel to the flavin isoalloxazine ring, but lay against it at angles of ～45° and ～35°, respectively. The adenine ribose moiety of NADH was surrounded by loop ?2 on chain B and α3 on chain A in a typical Rossmann fold. There were 12 and 19 amino acid residues that could participate in the binding of Methyl Red and NADH, respectively, especially the residues Tyr-129 and Asp-184. The functional perturbation effects of 13 residues, including Tyr-129 and Asp-184, were mapped to reconstruct the mutation network, which confirmed the proposed binding modes and also provided insights into the interaction among NADH, FMN and Methyl Red.     

Molecular cloning, overexpression, purification, and characterization of an aerobic FMN-dependent azoreductase from Enterococcus faecalis

Azo dyes represent a major class of synthetic colorants that are ubiquitous in foods and consumer products. Enterococcus faecalis is a predominant member of the human gastrointestinal microflora. Strain ATCC 19433 grew in the presence of azo dyes and metabolized them to colorless products. A gene encoding a putative FMN-dependent aerobic azoreductase that shares 34% identity with azoreductase (AcpD) of Escherichia coli has been identified in this strain. The gene in E. faecalis, designated as azoA, encoded a protein of 208 amino acids with a calculated isoelectric point of 4.8. AzoA was heterologously overexpressed in E. coli with a strong band of 23 kDa on SDS-PAGE. The purified recombinant enzyme was a homodimer with a molecular weight of 43 kDa, probably containing one molecule of FMN per dimer. AzoA required FMN and NADH, but not NADPH, as a preferred electron donor for its activity. The apparent Km values for both NADH and 2-[4-(dimethylamino)phenylazo]benzoic acid (Methyl red) substrates were 0.14 and 0.024 mM, respectively. The apparent Vmax was 86.2 microM/min/mg protein. The enzyme was not only able to decolorize Methyl red, but was also able to convert sulfonated azo dyes Orange II, Amaranth, Ponceau BS, and Ponceau S. AzoA is the first aerobic azoreductase to be identified and characterized from human intestinal gram-positive bacteria.     

Mutation network-based understanding of pleiotropic and epistatic mutational behavior of Enterococcus faecalis FMN-dependent azoreductase

We previously identified a highly active homodimeric FMN-dependent NADH-preferred azoreductase (AzoA) from Enterococcus faecalis, which cleaves the azo bonds (R-N?N-R) of diverse azo dyes, and determined its crystal structure. The preliminary network-based mutational analysis suggested that the two residues, Arg-21 and Asn-121, have an apparent mutational potential for fine-tuning of AzoA, based on their beneficial pleiotropic feedbacks. However, epistasis between the two promising mutational spots in AzoA has not been obtained in terms of substrate binding and azoreductase activity. In this study, we further quantified, visualized, and described the pleiotropic and/or epistatic behavior of six single or double mutations at the positions, Arg-21 and Asn-121, as a further research endeavor for beneficial fine-tuning of AzoA. Based on this network-based mutational analysis, we showed that pleiotropy and epistasis are common, sensitive, and complex mutational behaviors, depending mainly on the structural and functional responsibility and the physicochemical properties of the residue(s) in AzoA.     

Purification and characterization of an oxygen-insensitive NAD(P)H nitroreductase from Enterobacter cloacae

The reductive products of several nitroaromatic compounds have been found to be toxic, mutagenic, and carcinogenic. The nitroreductases present in intestinal microflora have been implicated in the biotransformation of these compounds to their deleterious metabolites. A "classical" nitroreductase has been purified from Enterobacter cloacae 587-fold using a protocol which yields approximately 1 mg of purified nitroreductase from 10 liters of cell culture. An analysis of the physical properties of the nitroreductase indicates that the enzyme is active as a monomer with a calculated molecular mass of 27 kDa. FMN has been identified as a required flavin cofactor and is present at a stoichiometry of 0.88 mol of FMN bound/mol of active enzyme. The enzyme was found capable of reducing nitrofurazone under aerobic conditions indicating that the mechanism involves an obligatory two-electron transfer. Thus, this enzyme can be classified as an oxygen-insensitive nitroreductase. The purified nitroreductase can utilize either NADH or NADPH as a source of reducing equivalents and can reduce a variety of nitroaromatic compounds including nitrofurans and nitrobenzenes as well as quinones. Studies in which the rates of nitroreduction for a series of para substituted nitrobenzene derivatives were determined suggest that a linear free energy relationship exists between the rate and the redox midpoint potential of the substrate.     

Purification and Characterization of an NAD(P)H:Quinone Oxidoreductase from Glycine Max Seedlings

Abstract

An NAD(P)H:(quinone acceptor) oxidoreductase (EC 1.6.99.2) was purified from Glycine max seedlings by means of chromatographic procedures. After 1371-fold purification, the enzyme showed a single band in IEF corresponding to an isoelectric point of 6.1. A single band was also found in native-PAGE both by activity staining and Coomassie brilliant blue staining. The molecular mass determined in SDS-PAGE was 21900 Da, while in HPLC gel-filtration it was 61000 Da. The NAD(P)H:quinone oxidoreductase was able to use NADH or NADPH as the electron donor. Among the artificial quinones which are reduced by this enzyme, 6-hydroxydopa- and 6-hydroxydopamine-quinone are of particular interest because of their neurotoxic effects.     

Solubilization and Purification of NAD(P)H Dehydrogenase of Cucurbita Microsomes

An NAD(P)H dehydrogenase stimulated by quinone (P Pupillo, V Valenti, L de Luca, R Hertel 1986 Plant Physiol 80: 384-389) was solubilized from washed microsomes of zucchini squash hypocotyls (Cucurbita pepo L.) by use of 1% Triton X-100. The solubilized enzyme remained in solution in aqueous buffer and could be purified by a combination of Sepharose 6B chromatography and Blue Ultrogel chromatography. Of the three peaks of activity eluted from the latter column with a salt gradient, peak 3 had 50% or more of the activity and was almost pure enzyme. The preparation examined in SDS-gel electrophoresis consisted of two types of subunits, a (molecular weight 39,500) and b (37,000) in equal amounts. Peak 2 was less pure but had a similar polypeptide pattern. The active protein is proposed to be a heterotetramer (a₂b₂) having a molecular weight of about 150,000, as found by gel exclusion chromatography. The purified enzyme can reduce several quinones, DCPIP, cytochrome c, and with best efficiency ferricyanide, and is therefore a diaphorase. The kinetics for the substrates are negatively cooperative with Hill coefficients n_H = 0.55 ± 0.05 for NADPH and 0.22 ± 0.04 for duroquinone. A weak inhibition by p-hydroxymercuric benzoate and mersalyl (stronger with microsomal preparations) suggests the presence of essential sulfhydryl group(s). The possibility is discussed that the dehydrogenase is an NAD(P)H-P450 reductase or similar flavoprotein, and that it is responsible for the NADPH-cytochrome c reductase activity of plant microsomes.     

NAD(P)H dehydrogenase from rabbit and rat liver: Purification and some properties

• 1.1. NAD(P)H dehydrogenase from rabbit liver was purified to electrophoretic homogeneity using a procedure also found applicable for the rat liver enzyme.
• 2.2. Rabbit and rat liver enzymes showed different behaviour in isoelectric focusing and different K_m values and turnover numbers.
• 3.3. Both enzymes were inhibited to similar extents by warfarin.
• 4.4. The rabbit enzyme is composed of two subunits of mol. wt 27,000 and contained 1 FAD group per subunit.
• 5.5. Some absorption and circular dichroism properties of the rat enzyme are shown.

     

Partial Purification and Characterization of Three NAD(P)H Dehydrogenases from Beta vulgaris Mitochondria

Mitochondria isolated from the taproot of beet (Beta vulgaris) were used in an effort to identify and partially purify the proteins constituting the exogenous NADH dehydrogenase. Three NAD(P)H dehydrogenases are released from these mitochondria by sonication, and these enzymes were partially purified using fast protein liquid chromatography. One of the enzymes, designated peak I, is capable of oxidizing NADPH and the β form of NADH. The other two activities, peaks II and III, oxidize only β-NADH. All three peaks are insensitive to divalent cation chelators and a complex I inhibitor, rotenone. The major component to peak I is a polypeptide with an apparent molecular mass of approximately 42 kilodaltons. Peak I activity was insensitive to platanetin, a specific inhibitor of the exogenous dehydrogenase, and insensitive to added Ca²⁺ or Mg²⁺. Peak I displayed a broad pH activity profile with an optimum between 7.5 and 8.0 for both NADPH and NADH. Purified peak II gave a single polypeptide of about 32 kilodaltons, had a pH optimum between 7.0 and 7.5, and was slightly stimulated by Ca²⁺ and Mg²⁺. As with peak I, platanetin had no effect on peak II activity. Peak III was not purified completely, but contained two major polypeptides with apparent molecular masses of 55 and 40 kilodaltons. This enzyme was not affected by Ca²⁺ and Mg²⁺, but was inhibited by platanetin. The peak III enzyme had a rather sharp pH optimum of approximately 6.5 to 6.6. The above data indicate that peak III activity is likely the exogenous NADH dehydrogenase.     

Purification and Partial Characterization of Two Soluble NAD(P)H Dehydrogenases from Arum maculatum Mitochondria

Two enzyme systems carrying out the oxidation of NAD(P)H in the presence of various electron acceptors have been isolated and partially characterized from the supernatant of frozen-thawed mitochondria from Arum maculatum spadices. The two systems contain flavoproteins and differ by their ability to oxidize NADH or NADPH, optimum pH and pI values, sensitivity to Ca²⁺ and EGTA, denaturation by 4 molar urea, molecular mass, and number of subunits. These properties, together with methodological considerations, are compatible with the location of these enzyme activities on the outer surface of the inner mitochondrial membrane, and support the hypothesis of the existence of two separate dehydrogenases responsible for the mitochondrial oxidation of cytosolic NADH and NADPH.     

Effect of culture conditions on the NADH/NAD ratio and total amounts of NAD(H) in chemostat cultures of Enterococcus faecalis NCTC 775

Abstract Enterococcus faecalis was grown in chemostat culture on various energy sources at dilution rates ranging from 0.05 h⁻¹ to 0.5 h⁻¹, under both aerobic and anaerobic conditions. NADH/NAD ratios and total nicotinamide adenine dinucleotide pool size (NAD(H)) were determined. It was found that the NADH/NAD ratio was controlled by the steady state product concentrations rather than by the degree of reduction of the energy source. Highest ratios were observed when NADH was reoxidized via ethanol formation, whereas in aerobic cultures, in which predominantly acetate was produced and oxidation of NADH occurred via the NADH oxidase, ratios were lowest. Addition of ethanol to the medium resulted in an increase of the NADH/NAD ratio, both aerobically and anaerobically. The total amount of NAD(H) was found to be influenced by the culture conditions. Under anaerobic conditions, the NADH oxidation (NAD reduction) rate appeared to correlate with the total amount of nicotinamide nucleotides. In contrast, no effect of the culture conditions on the total amount of NAD(H) was observed in aerobically grown cells.     

Vanadate-stimulated oxidation of NAD(P)H

Vanadate stimulates the oxidation of NAD(P)H by biological membranes because such membranes contain NAD(P)H oxidases which are capable of reducing dioxygen to O₂⁻ and because vanadate catalyzes the oxidation of NAD(P)H by O₂⁻, by a free radical chain mechanism. Dihydropyridines, such as reduced nicotinamide mononucleotide (NMNH), which are not substrates for membrane-associated NAD(P)H oxidases, are not oxidized by membranes plus vanadate unless NAD(P)H is present to serve as a source of O₂⁻. When [NMNH] greatly exceeds [NAD(P)H], in such reaction mixtures, one can observe the oxidation of many molecules of NMNH per NAD(P)H consumed. This reflects the chain length of the free radical chain mechanism. We have discussed the mechanism and significance of this process and have tried to clarify the pertinent but confusing literature.     

Characterization of thermostable FMN-dependent NADH azoreductase from the moderate thermophile Geobacillus stearothermophilus

The gene encoding an FMN-dependent NADH azoreductase, AzrG, from thermophilic Geobacillus stearothermophilus was cloned and functionally expressed in recombinant Escherichia coli. Purified recombinant AzrG is a homodimer of 23 kDa and bore FMN as a flavin cofactor. The optimal temperature of AzrG was 85 °C for the degradation of Methyl Red (MR). AzrG remained active for 1 h at 65 °C and for 1 month at 30 °C, demonstrating both superior thermostability and long-term stability of the enzyme. AzrG efficiently decolorized MR, Ethyl Red at 30 °C. Furthermore, the enzyme exhibited a wide-range of degrading activity towards several tenacious azo dyes, such as Acid Red 88, Orange I, and Congo Red. These results suggested the sustainable utilization of G. stearothermophilus as an azo-degrading strain for AzrG carrying whole-cell wastewater treatments for azo pollutants under high temperature conditions.     

Identification and Characterization of an Inducible NAD(P)H Dehydrogenase from Red Beetroot Mitochondria

Exogenous NADH oxidation of mitochondria isolated from red beetroots (Beta vulgaris L.) increased dramatically upon slicing and aging the tissue. Anion-exchange chromatography of soluble fractions derived by sonication from fresh and aged beetroot mitochondria yielded three NADH dehydrogenase activity peaks. The third peak from aged beetroot mitochondria was separated into two activities by blue-affinity chromatography. One of these (the unbound peak) readily oxidized dihydrolipoamide, whereas the other (the bound peak) did not. The latter was an NAD(P)H dehydrogenase with high quinone and ferricyanide reductase activity and was absent from fresh beet mitochondria. Further affinity chromatography of the NAD(P)H dehydrogenase indicated enrichment of a 58-kD polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We propose that this 58-kD protein is the inducible, external NADH dehydrogenase.     

Purification and characterization of two isofunctional forms of NAD(P)H: quinone reductase from mouse liver

NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2) is a widely distributed enzyme which promotes two-electron reductions of quinones and thereby protects cells against damage by reactive oxygen species generated during oxidative cycling of quinones and semiquinone radicals. Quinone reductase activity represents a minor component (about 0.006%) of mouse liver cytosolic proteins under basal (uninduced) conditions. Two isofunctional forms of this quinone reductase have been purified to homogeneity (1700-fold) in 30% yield from the liver cytosols of female CD-1 mice in which the enzymes were induced by administration of 2(3)-tert-butyl-4-hydroxyanisole. The purification involved ion exchange, hydrophobic, and affinity chromatographies. The two enzyme forms have been designated "hydrophilic" and "hydrophobic" based on the order of elution from phenyl-Sepharose. The more abundant hydrophilic form has been crystallized in the presence of FAD in the form of macroscopic tetragonal crystals. The two forms have similar isoelectric points (pI 9.2) and subunit molecular weights (Mr = 30,000) and probably exist as dimers in the native state. Purified preparations of the enzymes are equiactive with NADH and NADPH and show almost complete dependence on added FAD for catalytic activity. The Km values for FAD of the hydrophilic and hydrophobic forms are 2.72 and 1.72 nM, respectively. Their catalytic activities are the same and are remarkably high for nicotinamide nucleotide-linked dehydrogenases; maximum velocities (expressed per mg of pure enzyme) approach 4000 units/mg of protein under appropriate assay conditions. When menadione is the electron acceptor, the Km value for this quinone is very low (Km congruent to 2 microM). Both enzyme forms are potently inhibited by dicoumarol. Rabbit antisera against the hydrophilic quinone reductase precipitate quantitatively the entire quinone reductase activity of mouse liver cytosols obtained from animals maintained on a standard diet or those induced with 3-tert-butyl-4-hydroxyanisole. The quinone reductase activity of rat liver cytosols is also quantitatively precipitated by this antiserum.     

Preliminary X-ray studies on an unspecific NAD(P)H dehydrogenase from human erythrocytes

Crystals of a relatively unspecific NAD(P)H dehydrogenase from human erythrocytes suitable for X-ray analysis have been grown. They belong to space group P4₁2₁2 or its enantiomorph P4₃2₁2 with unit cell dimensions: $\text{a = b = 79.3}\text{A}\text{?}\text{and}\text{c = 38.1}\text{A}\text{?}$. The asymmetric unit contains one molecule of M_r 18,000.     

Purification and Characterization of NAD(P)H:Nitrate Reductase and NADH:Nitrate Reductase from Corn Roots

The nitrate reductase activity of 5-day-old whole corn roots was isolated using phosphate buffer. The relatively stable nitrate reductase extract can be separated into three fractions using affinity chromatography on blue-Sepharose. The first fraction, eluted with NADPH, reduces nearly equal amounts of nitrate with either NADPH or NADH. A subsequent elution with NADH yields a nitrate reductase which is more active with NADH as electron donor. Further elution with salt gives a nitrate reductase fraction which is active with both NADH and NADPH, but is more active with NADH. All three nitrate reductase fractions have pH optima of 7.5 and Stokes radii of about 6.0 nanometers. The NADPH-eluted enzyme has a nitrate K_m of 0.3 millimolar in the presence of NADPH, whereas the NADH-eluted enzyme has a nitrate K_m of 0.07 millimolar in the presence of NADH. The NADPH-eluted fraction appears to be similar to the NAD(P)H:nitrate reductase isolated from corn scutellum and the NADH-eluted fraction is similar to the NADH:nitrate reductases isolated from corn leaf and scutellum. The salt-eluted fraction appears to be a mixture of NAD(P)H: and NADH:nitrate reductases.     

Purification and some properties of NAD(P)H dehydrogenase from Saccharomyces cerevisiae: Contribution to glycolytic methylglyoxal pathway

NAD(P)H dehydrogenase was purified approximately 480-fold from Saccharomyces cerevisiae with 6.5% activity yield. The enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 40,000–44,000 by gel filtration on Sephadex G-150 column chromatography and SDS-polyacrylamide gel electrophoresis. The K_m values for NADPH and NADH were 7.3 μM and 0.1 mM, respectively. The activity of the enzyme increased approximately 4-fold with Cu²⁺. FAD, FMN and cytochrome c were not effective as electron acceptors, although Fe(CN)₆³⁻ was slightly effective. NADH generated by the reaction of lactaldehyde dehydrogenase in the glycolytic methylglyoxal pathway will be reoxidized by NAD(P)H dehydrogenase. NAD(P)H dehydrogenase thus may contribute to the reduction/oxidation system in the glycolytic methylglyoxal pathway to maintain the flux of methylglyoxal to lactic acid via lactaldehyde.     

Biphasic oxidation of mitochondrial NAD(P)H

The redox state of mitochondrial pyridine nucleotides is known to be important for structural integrity of mitochondria. In this work, we observed a biphasic oxidation of endogenous NAD(P)H in rat liver mitochondria induced by tert-butylhydroperoxide. Nearly 85% of mitochondrial NAD(P)H was rapidly oxidized during the first phase. The second phase of NAD(P)H oxidation was retarded for several minutes, appearing after the inner membrane potential collapse and mitochondria swelling. It was characterized by disturbance of ATP synthesis and dramatic permeabilization of the inner membrane to pyridine nucleotides. The second phase was completely prevented by 0.5 microM cyclosporin A or 0.2 mM EGTA or was significantly delayed by 25 microM butylhydroxytoluene or trifluoperazine. The obtained data suggest that the second phase resulted from oxidation of the remaining NADH via the outer membrane electron transport system of permeabilized mitochondria, leading to further oxidation of the remaining NADPH in a transhydrogenase reaction.     

Purification,Characterization, and Submitochondrial Localization of a 58-Kilodalton NAD(P)H Dehydrogenase

An NADH dehydrogenase activity from red beet (Beta vulgaris L.) root mitochondria was purified to a 58-kD protein doublet. An immunologically related dehydrogenase was partially purified from maize (Zea mays L. B73) mitochondria to a 58-kD protein doublet, a 45-kD protein, and a few other less prevalent proteins. Polyclonal antibodies prepared against the 58-kD protein of red beet roots were found to immunoprecipitate the NAD(P)H dehydrogenase activity. The antibodies cross-reacted to similar proteins in mitochondria from a number of plant species but not to rat liver mitochondrial proteins. The polyclonal antibodies were used in conjunction with maize mitochondrial fractionation to show that the 58-kD protein was likely part of a protein complex loosely associated with the membrane fraction. A membrane-impermeable protein cross-linking agent was used to further show that the majority of the 58-kD protein was located on the outer surface of the inner mitochondrial membrane or in the intermembrane space. Analysis of the cross-linked 58-kD NAD(P)H dehydrogenase indicated that specific proteins of 64, 48, and 45 kD were cross-linked to the 58-kD protein doublet. The NAD(P)H dehydrogenase activity was not affected by ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime] -tetraacetic acid or CaCl2, was stimulated somewhat (21%) by flavin mononucleotide, was inhibited by p-chloromercuribenzoic acid (49%) and mersalyl (40%), and was inhibited by a bud scale extract of Platanus occidentalis L. containing platanetin (61%).