Modeled structure of trypanothione reductase of Leishmania infantum

Trypanothione reductase is an important target enzyme for structure-based drug design against Leishmania. We used homology modeling to construct a three-dimensional structure of the trypanothione reductase (TR) of Leishmania infantum. The structure shows acceptable Ramachandran statistics and a remarkably different active site from glutathione reductase(GR). Thus, a specific inhibitor against TR can be designed without interfering with host (human) GR activity.     

Development of cell-free protein synthesis platforms for disulfide bonded proteins

The use of cell-free protein synthesis (CFPS) for recombinant protein production is emerging as an important technology. For example, the openness of the cell-free system allows control of the reaction environment to promote folding of disulfide bonded proteins in a rapid and economically feasible format. These advantages make cell-free protein expression systems particularly well suited for producing patient specific therapeutic vaccines or antidotes in response to threats from natural and man-made biological agents and for pharmaceutical proteins that are difficult to produce in living cells. In this work we assess the versatility of modern cell-free methods, optimize expression and folding parameters, and highlight the importance of rationally designed plasmid templates for producing mammalian secreted proteins, fusion proteins, and antibody fragments in our E. coli-based CFPS system. Two unique CFPS platforms were established by developing standardized extract preparation protocols and generic cell-free reaction conditions. Generic reaction conditions enabled all proteins to express well with the best therapeutic protein yield at 710 microg/mL, an antibody fragment at 230 microg/mL, and a vaccine fusion protein at 300 microg/mL; with the majority correctly folded. Better yields were obtained when cell-free reaction conditions were optimized for each protein. Establishing general CFPS platforms enhances the potential for cell-free protein synthesis to reliably produce complex protein products at low production and capital costs with very rapid process development timelines.     

Flavin adenine dinucleotide content of quinone reductase 2: analysis and optimization for structure-function studies

Quinone reductase 2 (NQO2) is a broadly expressed enzyme implicated in responses to a number of compounds, including protein kinase inhibitors, resveratrol, and antimalarial drugs. NQO2 includes a flavin adenine dinucleotide (FAD) cofactor, but X-ray crystallographic analysis of human NQO2 expressed in Escherichia coli showed that electron density for the isoalloxazine ring of FAD was weak and there was no electron density for the adenine mononucleotide moiety. Reversed-phase high-performance liquid chromatography (HPLC) of the NQO2 preparation indicated that FAD was not present and only 38% of the protomers contained flavin mononucleotide (FMN), explaining the weak electron density for FAD in the crystallographic analysis. A method for purifying NQO2 and reconstituting with FAD such that the final content approaches 100% occupancy with FAD is presented here. The enzyme prepared in this manner has a high specific activity, and there is strong electron density for the FAD cofactor in the crystal structure. Analysis of NQO2 crystal structures present in the Protein Data Bank indicates that many may have sub-stoichiometric cofactor content and/or contain FMN rather than FAD. This method of purification and reconstitution will help to optimize structural and functional studies of NQO2 and possibly other flavoproteins.     

Docking and molecular dynamics studies at trypanothione reductase and glutathione reductase active sites

A theoretical docking study on the active sites of trypanothione reductase (TR) and glutathione reductase (GR) with the corresponding natural substrates, trypanothione disulfide (T[S]2) and glutathione disulfide (GSSG), is reported. Molecular dynamics simulations were carried out in order to check the robustness of the docking results. The energetic results are in agreement with previous experimental findings and show the crossed complexes have lower stabilization energies than the natural ones. To test DOCK3.5, four nitro furanic compounds, previously designed as potentially active anti-chagasic molecules, were docked at the GR and TR active sites with the DOCK3.5 procedure. A good correlation was found between differential inhibitory activity and relative interaction energy (affinity). The results provide a validation test for the use of DOCK3.5 in connection with the design of anti-chagasic drugs.     

Arylfurans as potential trypanosoma cruzi trypanothione reductase inhibitors

The natural lignans veraguensin and grandisin have been reported to be active against Trypanosoma cruzi bloodstream forms. Aiming at the total synthesis of these and related compounds, we prepared three 2-arylfurans and eight 2,5-diarylfurans. They were evaluated for their potential as T. cruzi trypanothione reductase (TR) inhibitors as well against the parasite's intracellular (amastigote) and bloodstream (trypomastigote) forms. Compound 12 was the most effective against TR with an IC50 of 48.5 microM while 7 and 14 were active against amastigotes, inhibiting the parasite development by 60% at 20 microg/ml (59 and 90 microM, respectively). On the other hand, none of the compounds was significantly active against the parasite bloodstream forms even at 250 microg/ml (0.6-1.5 mM).     

Electron-transfer reactions of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath).

Soluble methane monooxygenase (sMMO) catalyzes the hydroxylation of methane by dioxygen to afford methanol and water, the first step of carbon assimilation in methanotrophic bacteria. This enzyme comprises three protein components: a hydroxylase (MMOH) that contains a dinuclear nonheme iron active site; a reductase (MMOR) that facilitates electron transfer from NADH to the diiron site of MMOH; and a coupling protein (MMOB). MMOR uses a noncovalently bound FAD cofactor and a [2Fe-2S] cluster to mediate electron transfer. The gene encoding MMOR was cloned from Methylococcus capsulatus (Bath) and expressed in Escherichia coli in high yield. Purified recombinant MMOR was indistinguishable from the native protein in all aspects examined, including activity, mass, cofactor content, and EPR spectrum of the [2Fe-2S] cluster. Redox potentials for the FAD and [2Fe-2S] cofactors, determined by reductive titrations in the presence of indicator dyes, are FAD(ox/sq), -176 +/- 7 mV; FAD(sq/hq), -266 +/- 15 mV; and [2Fe-2S](ox/red), -209 +/- 14 mV. The midpoint potentials of MMOR are not altered by the addition of MMOH, MMOB, or both MMOH and MMOB. The reaction of MMOR with NADH was investigated by stopped-flow UV-visible spectroscopy, and the kinetic and spectral properties of intermediates are described. The effects of pH on the redox properties of MMOR are described and exploited in pH jump kinetic studies to measure the rate constant of 130 +/- 17 s(-)(1) for electron transfer between the FAD and [2Fe-2S] cofactors in two-electron-reduced MMOR. The thermodynamic and kinetic parameters determined significantly extend our understanding of the sMMO system.     

Kinetic and structural properties of biocatalytically active sheep lung microsomal NADPH-cytochrome c reductase

NADPH-cytochrome c reductase was purified to electrophoretic homogeneity from detergent solubilized sheep lung microsomes. The specific activity of the purified enzyme ranged from 56 to 67 mumol cytochrome c reduced/min/mg protein and the yield was 48-52% of the initial activity in lung microsomes. The reductase had Mr of 78,000 and contained 1 mol each of FAD and FMN. Km values obtained in 0.3 M phosphate buffer, pH 7.8 at 37 degrees C for NADPH and cytochrome c were 11.1 +/- 0.70 microM and 20.0 +/- 2.15 microM. Lung reductase was inhibited by its substrate, cytochrome c when its concentration was above 160 microM. The lung reductase exhibited a ping-pong type kinetic mechanism for NADPH mediated cytochrome c reduction. Purified lung reductase was biocatalytically active in supporting benzo(a)pyrene hydroxylation reaction when coupled with lung cytochrome P-450 and lipid.     

Deconstructing the electron transfer chain in a complex molybdoenzyme: Assimilatory nitrate reductase from Neurospora crassa

Nitrate reductase (NR) from the fungus Neurospora crassa is a complex homodimeric metallo-flavoenzyme, where each protomer contains three distinct domains; the catalytically active terminal molybdopterin cofactor, a central heme-containing domain, and an FAD domain which binds with the natural electron donor NADPH. Here, we demonstrate the catalytic voltammetry of variants of N. crassa NRs on a modified Au electrode with the electrochemically reduced forms of benzyl viologen (BV²⁺) and anthraquinone sulfonate (AQS^?) acting as artificial electron donors. The biopolymer chitosan used to entrap NR on the electrode non-covalently and the enzyme film was both stable and highly active. Electrochemistry was conducted on two distinct forms; one lacking the FAD cofactor and the other lacking both the FAD and heme cofactors. While both enzymes showed catalytic nitrate reductase activity, removal of the heme cofactor resulted in a more significant effect on the rate of nitrate reduction. Electrochemical simulation was carried out to enable kinetic characterisation of both the NR:nitrate and NR:mediator reactions.     

Catalysis in fumarate reductase

In the absence of oxygen many bacteria are able to utilise fumarate as a terminal oxidant for respiration. In most known organisms the fumarate reductases are membrane-bound iron-sulfur flavoproteins but Shewanella species produce a soluble, periplasmic flavocytochrome c(3) that catalyses this reaction. The active sites of all fumarate reductases are clearly conserved at the structural level, indicating a common mechanism. The structures of fumarate reductases from two Shewanella species have been determined. Fumarate, succinate and a partially hydrated fumarate ligand are found in equivalent locations in different crystals, tightly bound in the active site and close to N5 of the FAD cofactor, allowing identification of amino acid residues that are involved in substrate binding and catalysis. Conversion of fumarate to succinate requires hydride transfer from FAD and protonation by an active site acid. The identity of the proton donor has been open to question but we have used structural considerations to suggest that this function is provided by an arginine side chain. We have confirmed this experimentally by analysing the effects of site-directed mutations on enzyme activity. Substitutions of Arg402 lead to a dramatic loss of activity whereas neither of the two active site histidine residues is required for catalysis.     

Effect of dinitrophenyl modification on oxidation-reduction of glutathione reductase from yeast

Covalent modification of glutathione reductase (GR) from yeast with 1-fluoro-2,4-dinitrobenzene (FDNB) inhibited the NADPH-GSSG reductase activity completely. This modification also decreased the NADPH-thio-NADP+ transhydrogenase activity, stimulated the NADPH-oxidase activity, and induced the NADPH-cytochrome c reductase activity. Spectrophotometric titration showed that one tyrosine residue per FAD was modified with a dinitrophenyl group. The modified enzyme showed conversion of the two-electron reduced form (EH2) to the four-electron reduced form (EH4) in anaerobic conditions and conversion of EH2 to the oxidized form (E) in aerobic conditions. These results indicate that the modification of one tyrosine residue of the active site induces the instability of EH2.     

Flavin specificity and subunit interaction of Vibrio fischeri general NAD(P)H-flavin oxidoreductase FRG/FRase I

Apoenzyme of the major NAD(P)H-utilizing flavin reductase FRG/FRase I from Vibrio fischeri was prepared. The apoenzyme bound one FMN cofactor per enzyme monomer to yield fully active holoenzyme. The FMN cofactor binding resulted in substantial quenching of both the flavin and the protein fluorescence intensities without any significant shifts in the emission peaks. In addition to FMN binding (K(d) 0.5 microM at 23 degrees C), the apoenzyme also bound 2-thioFMN, FAD and riboflavin as a cofactor with K(d) values of 1, 12, and 37 microM, respectively, at 23 degrees C. The 2-thioFMN containing holoenzyme was about 40% active in specific activity as compared to the FMN-containing holoenzyme. The FAD- and riboflavin-reconstituted holoenzymes were also catalytically active but their specific activities were not determined. FRG/FRase I followed a ping-pong kinetic mechanism. It is proposed that the enzyme-bound FMN cofactor shuttles between the oxidized and the reduced form during catalysis. For both the FMN- and 2-thioFMN-containing holoenzymes, 2-thioFMN was about 30% active as compared to FMN as a substrate. FAD and riboflavin were also active substrates. FRG/FRase I was shown by ultracentrifugation at 4 degrees C to undergo a monomer-dimer equilibrium, with K(d) values of 18.0 and 13.4 microM for the apo- and holoenzymes, respectively. All the spectral, ligand equilibrium binding, and kinetic properties described above are most likely associated with the monomeric species of FRG/FRase I. Many aspects of these properties are compared with a structurally and functionally related Vibrio harveyi NADPH-specific flavin reductase FRP.     

Response of plasma and red cell glutathione reductase of several mammalian species and domestic chickens to flavin adenine dinucleotide.

Glutathione reductase (GR) activity was determined in the plasma and red cells from a number of species (human, horse, cattle, sheep, chicken, rabbit, rat). The species with the highest plasma activity (horse and cattle) had the lowest red cell activity. Unlike red cell GR, plasma GR was not significantly stimulated by the addition of flavin adenine dinucleotide (FAD). In species with high plasma GR activity the determination of whole blood FAD activity coefficients may not be a good index of riboflavin status.     

Flavin fluorescence dynamics and photoinduced electron transfer in Escherichia coli glutathione reductase.

Time-resolved polarized flavin fluorescence was used to study the active site dynamics of Escherichia coli glutathione reductase (GR). Special consideration was given to the role of Tyr177, which blocks the access to the NADPH binding-site in the crystal structure of the enzyme. By comparing wild-type GR with the mutant enzymes Y177F and Y177G, a fluorescence lifetime of 7 ps that accounts for approximately 90% of the fluorescence decay could be attributed to quenching by Y177. Based on the temperature invariance for this lifetime, and the very high quenching rate, electron transfer from Y177 to the light-excited isoalloxazine part of flavin adenine dinucleotide (FAD) is proposed as the mechanism of flavin fluorescence quenching. Contrary to the mutant enzymes, wild-type GR shows a rapid fluorescence depolarization. This depolarization process is likely to originate from a transient charge transfer interaction between Y177 and the light-excited FAD, and not from internal mobility of the flavin, as has previously been proposed. Based on the fluorescence lifetime distributions, the mutants Y177F and Y177G have a more flexible protein structure than wild-type GR: in the range of 223 K to 277 K in 80% glycerol, both tyrosine mutants mimic the closely related enzyme dihydrolipoyl dehydrogenase. The fluorescence intensity decays of the GR enzymes can only be explained by the existence of multiple quenching sites in the protein. Although structural fluctuations are likely to contribute to the nonexponential decay and the probability of quenching by a specific site, the concept of conformational substates need not be invoked to explain the heterogeneous fluorescence dynamics.     

Population study of erythrocyte glutathione reductase activity

Summary The activity of erythrocyte glutathione reductase (GR) was determined in a group of 87 prisoners from northern Thailand (65 with normal, 22 with deficient erythrocyte G-6-PD) without and with added FAD. The amount of stimulation by FAD was inversely related to the original activity suggesting that FAD stimulation in vivo is one of the main determinants of GR activity. 4 subjects showed insufficient stimulation by FAD. The binding of FAD to GR seemed to be closer to saturation in G-6-PD deficient subjects but the maximal stimulated activity of these subjects was higher than in the group with normal G-6-PD. This suggests that the marked increase of GR activity in G-6-PD deficient erythrocytes is due to increased binding of FAD and to a larger amount of stimulable enzyme. Original GR activity was positively correlated with the socio-economic status, and subjects who had taken riboflavin in the period prior to examination had a higher mean GR activity than those without vitamin intake. GR activity was markedly raised by administration of riboflavin and in G-6-PD deficient subjects glutathione stability was improved. In comparison to central European subjects more than 50 percent of the examined population are GR deficient. This seems to be due to a low dietary supply of riboflavin in most cases. The findings in 4 subjects with insufficient stimulation by added FAD raise the question whether hereditary forms of GR deficiency exist in this population.

---

Zusammenfassung In einer Gruppe von 87 Strafgefangenen in Nordthailand (65 mit normaler, 22 mit defizienter G-6-PD) wurde die Aktivität der Glutathion-Reductase (GR) mit und ohne Zusatz von FAD bestimmt. Das Ausmaß der Stimulierung des Enzyms durch FAD verhielt sich reziprok zur ausgangsaktivität. Bei 4 Versuchspersonen war die Stimulierung durch FAD insuffizient. Die Bindung von FAD an GR war bei Versuchspersonen mit G-6-PD-Mangel näher am Sättigungspunkt. Aber auch die gesamte stimulierbare Aktivität der GR war bei Versuchspersonen mit G-6-PD-Mangel größer als bei Gesunden. Die deutliche Erhöhung der GR-Aktivität bei G-6-PD-Mangel scheint zwei Ursachen zu haben: vermehrte Bindung von FAD an das Enzym und Vermehrung der Gesamtmenge an stimulierbarem Enzym. In einer weiteren Gruppe war die Ausgangsaktivität der GR vom sozio-ökonomischen Status der Versuchsperson abhängig. Personen, die vor der Untersuchung Riboflavin-haltige Medikamente eingenommen hatten, hatten höhere Aktivitäten. Die GR-Aktivität wurde durch Einnahme von Riboflavin unter Kontrolle stark erhöht, und bei Versuchspersonen mit G-6-PD-Mangel wurde die Glutathion-Stabilität der Erythrocyten verbessert. Im Vergleich zu Mitteleuropäern sind mehr als die Hälfte der thailändischen Versuchspersonen als GR defizient einzustufen. Dies ist wahrscheinlich auf eine geringere Zufuhr an Riboflavin in der Nahrung zurückzuführen. Die Befunde bei 4 Versuchspersonen mit insuffizienter Stimulierung der GR durch FAD könnte durch einen erblichen Mangel an GR verursacht sein.

---

---

Established and supported by Stiftung Volkswagenwerk.     

Rational design of peptide-based inhibitors of trypanothione reductase as potential antitrypanosomal drugs

Summary The rational design of ligands for the substrate-binding site of a homology-modelled trypanothione reductase (TR) was performed. Peptides were designed to be selective for TR over human glutathione reductase (GR). The design process capitalized on the proposed differences between the activesites of TR and human GR, subsequently confirmed by the TR crystal structure. Enzyme kinetics confirmed that forT. cruzi TR benzoyl-Leu-Arg-Arg-ß-naphthylamide was an inhibitor (K_i 13.8µM) linearly competitive with the native substrate, trypanothione disulphide, and did not inhibit glutathione reductase.     

Mycobacterium tuberculosis FprA, a novel bacterial NADPH-ferredoxin reductase.

The gene fprA of Mycobacterium tuberculosis, encoding a putative protein with 40% identity to mammalian adrenodoxin reductase, was expressed in Escherichia coli and the protein purified to homogeneity. The 50-kDa protein monomer contained one tightly bound FAD, whose fluorescence was fully quenched. FprA showed a low ferric reductase activity, whereas it was very active as a NAD(P)H diaphorase with dyes. Kinetic parameters were determined and the specificity constant (kcat/Km) for NADPH was two orders of magnitude larger than that of NADH. Enzyme full reduction, under anaerobiosis, could be achieved with a stoichiometric amount of either dithionite or NADH, but not with even large excess of NADPH. In enzyme titration with substoichiometric amounts of NADPH, only charge transfer species (FAD-NADPH and FADH2-NADP+) were formed. At NADPH/FAD ratios higher than one, the neutral FAD semiquinone accumulated, implying that the semiquinone was stabilized by NADPH binding. Stabilization of the one-electron reduced form of the enzyme may be instrumental for the physiological role of this mycobacterial flavoprotein. By several approaches, FprA was shown to be able to interact productively with [2Fe-2S] iron-sulfur proteins, either adrenodoxin or plant ferredoxin. More interestingly, kinetic parameters of the cytochrome c reductase reaction catalyzed by FprA in the presence of a 7Fe ferredoxin purified from M. smegmatis were determined. A Km value of 30 nm and a specificity constant of 110 microM(-1) x s(-1) (10 times greater than that for the 2Fe ferredoxin) were determined for this ferredoxin. The systematic name for FprA is therefore NADPH-ferredoxin oxidoreductase.     

The effects by neuroleptics, antimycotics and antibiotics on disulfide reducing enzymes from the human pathogens Acanthamoeba polyphaga and Naegleria fowleri

This paper discusses the effects of two neuroleptic agents, chlorpromazine and trifluoperazine; three antimycotics, amphotericin B, ketoconazole and miconazole and four antibiotics, pentamidine, rifampicin, mepacrine and metronidazole on the NADPH-dependent disulfide reducing enzymes cystine reductase (CysR), glutathione reductase (GR) trypanothione reductase (TR) and a putative disulfide reductase for compound X in Acanthamoeba polyphaga from the human pathogens A. polyphaga and Naegleria fowleri. Against A. polyphaga, all nine drugs studied had the capacity to inhibit the putative disulfide reductase from the trophozoites at a concentration of 32microg/ml during a 24h incubation and they were: the neuroleptics trifluoperazine (100%) and chlorpromazine (96%), the antimycotics miconazole (89%) ketoconazole (81%) and amphotericin B, (53%) and the antibiotics pentamidine (89%), rifampicin (64%), mepacrine (57%) and metronidazole (14%). Only six of the nine drugs simultaneously inhibited CysR, GR and the putative disulfide reductase. In N. fowleri, the most potent inhibitors of trypanothione reductase were amphotericin B and miconazole which inhibited 100% at a concentration of 32microg/ml during the 24h incubation followed by the neuroleptics trifluoperazine (92%) and chlorpromazine (80%) and the antibiotic mepacrine (70%). All these also inhibited CysR and GR from the trophozoites other than mepacrine which inhibited only CysR and TR. Ketoconazole, rifampicin (which did not affect CysR), pentamidine and metronidazole had opposite effects since they did not inhibit but increased the amount of the three thiols.