NADPH-dependent Disulfide Reductase Research Articles

New Insights Into the Function of Flavohemoglobin in Mycobacterium tuberculosis: Role as a NADPH-Dependent Disulfide Reductase and D-Lactate-Dependent Mycothione Reductase.

Mycobacterium tuberculosis (Mtb) produces an unconventional flavohemoglobin (MtbFHb) that carries a FAD-binding site similar to D-lactate dehydrogenases (D-LDH) and oxidizes D-lactate into pyruvate. The molecular mechanism by which MtbFHb functions in Mtb remains unknown. We discovered that the D-LDH-type FAD-binding site in MtbFHb overlaps with another FAD-binding motif similar to thioredoxin reductases and reduces DTNB in the presence of NADPH similar to trxB of Mtb. These results suggested that MtbFHb is functioning as a disulfide oxidoreductase. Interestingly, D-lactate created a conformational change in MtbFHb and attenuated its ability to oxidize NADPH. Mass spectroscopy demonstrated that MtbFHb reduces des-myo-inositol mycothiol in the presence of D-lactate unlike NADPH, indicating that D-lactate changes the specificity of MtbFHb from di-thiol to di-mycothiol. When M. smegmatis carrying deletion in the fhbII gene (encoding a homolog of MtbFHb) was complemented with the fhb gene of Mtb, it exhibited four- to fivefold reductions in lipid peroxidation and significant enhancement in the cell survival under oxidative stress. These results were corroborated by reduced lipid peroxidation and enhanced cell survival of wild-type M. smegmatis after overexpression of the fhb gene of Mtb. Since D-lactate is a by-product of lipid peroxidation and MtbFHb is a membrane-associated protein, D-lactate-mediated reduction of mycothiol disulfide by MtbFHb may uniquely equip Mtb to relieve the toxicity of D-lactate accumulation and protect the cell from oxidative damage, simultaneously balancing the redox environment under oxidative stress that may be vital for the pathogenesis of Mtb.

Disulfide reductase systems in liver.

Intermediary metabolism and detoxification place high demands on the disulfide reductase systems in most hepatocyte subcellular compartments. Biosynthetic, metabolic, cytoprotective and signalling activities in the cytosol; regulation of transcription in nuclei; respiration in mitochondria; and protein folding in endoplasmic reticulum all require resident disulfide reductase activities. In the cytosol, two NADPH-dependent enzymes, glutathione reductase and thioredoxin reductase, as well as a recently identified NADPH-independent system that uses catabolism of methionine to maintain pools of reduced glutathione, supply disulfide reducing power. However the necessary discontinuity between the cytosol and the interior of organelles restricts the ability of the cytosolic systems to support needs in other compartments. Maintenance of molecular- and charge-gradients across the inner-mitochondrial membrane, which is needed for oxidative phosphorylation, mandates that the matrix maintain an autonomous set of NADPH-dependent disulfide reductase systems. Elsewhere, complex mechanisms mediate the transfer of cytosolic reducing power into specific compartments. The redox needs in each compartment also differ, with the lumen of the endoplasmic reticulum, the mitochondrial inter-membrane space and some signalling proteins in the cytosol each requiring different levels of protein oxidation. Here, we present an overview of the current understanding of the disulfide reductase systems in major subcellular compartments of hepatocytes, integrating knowledge obtained from direct analyses on liver with inferences from other model systems. Additionally, we discuss relevant advances in the expanding field of redox signalling. LINKED ARTICLES: This article is part of a themed section on Chemical Biology of Reactive Sulfur Species. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.4/issuetoc.

1,4-Naphthoquinones and Other NADPH-Dependent Glutathione Reductase- Catalyzed Redox Cyclers as Antimalarial Agents

The homodimeric flavoenzyme glutathione reductase catalyzes NADPH-dependent glutathione disulfide reduction. This reaction is important for keeping the redox homeostasis in human cells and in the human pathogen Plasmodium falciparum. Different types of NADPH-dependent disulfide reductase inhibitors were designed in various chemical series to evaluate the impact of each inhibition mode on the propagation of the parasites. Against malaria parasites in cultures the most potent and specific effects were observed for redox-active agents acting as subversive substrates for both glutathione reductases of the Plasmodium-infected red blood cells. In their oxidized form, these redox-active compounds are reduced by NADPH-dependent flavoenzyme-catalyzed reactions in the cytosol of infected erythrocytes. In their reduced forms, these compounds can reduce molecular oxygen to reactive oxygen species, or reduce oxidants like methemoglobin, the major nutrient of the parasite, to indigestible hemoglobin. Furthermore, studies on a fluorinated suicide-substrate of the human glutathione reductase indicate that the glutathione reductase-catalyzed bioactivation of 3-benzylnaphthoquinones to the corresponding reduced 3-benzoyl metabolites is essential for the observed antimalarial activity. In conclusion, the antimalarial lead naphthoquinones are suggested to perturb the major redox equilibria of the targeted cells. These effects result in developmental arrest of the parasite and contribute to the removal of the parasitized erythrocytes by macrophages.

Characterization of a broad-range disulfide reductase from Streptomyces clavuligerus and its possible role in beta-lactam antibiotic biosynthesis.

Streptomyces clavuligerus is a potent producer of penicillin and cephalosporin antibiotics. A key step in the biosynthesis of these beta-lactam compounds is the cyclization of the thiol tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N by the enzyme isopenicillin N synthase (IPNS). However, bis-ACV, the oxidized disulfide form of the tripeptide, is not a substrate for IPNS. We show here that S. clavuligerus possesses an NADPH-dependent disulfide reductase of broad substrate specificity that efficiently catalyzes the reduction of disulfide bonds in bis-ACV and in other low-molecular-weight disulfide containing compounds and proteins. The disulfide reductase comprises two protein components, a 70-kDa reductase consisting of two identical subunits, and a 12-kDa heat-stable protein reductant. The structural and functional properties of the disulfide reductase resemble those of the thioredoxin class of oxidoreductases. When the disulfide reductase system is coupled with IPNS, it quantitatively converts bis-ACV to isopenicillin N. These findings suggest that the disulfide reductase may play a role in the biosynthesis of penicillins and cephalosporins in streptomycetes. We also show here that S. clavuligerus lacks glutathione reductase and have previously reported that Streptomyces species do not contain glutathione. This disulfide reductase may therefore be important in determining the thiol-disulfide redox balance in streptomycetes.

Biosynthesis of dimethyl selenide from sodium selenite in rat liver and kidney cell-free systems.

A pathway for the synthesis of dimethyl selenide from sodium selenite was studied in rat liver and kidney fractions under anaerobic conditions in the presence of GSH, a NADPH-generating system, and S-adenosylmethionine. Chromatography of liver or kidney soluble fraction on Sephadex G-75 yielded a Fraction C (30,000 molecular weight) which synthesized dimethyl selenide, but at a low rate. Addition of proteins eluting at the void volume (Fraction A) to Fraction C restored full activity. Fractionation of Fraction A on DEAE-cellulose revealed that its ability to stimulate Fraction C was associated with two fractions, one containing glutathione reductase and the other a NADPH-dependent disulfide reductase. It was concluded that Fraction C contains a methyltransferase acting on small amounts of hydrogen selenide produced non-enzymically by the reaction of selenite with GSH, and that stimulation by Fraction A results partly from the NADPH-linked formation of hydrogen selenide catalyzed by glutathione reductase present in Fraction A. Washed liver microsomal fraction incubated with selenite plus 20 mM GSH also synthesized dimethyl selenide, but addition of soluble fraction stimulated activity. A synergistic effect was obtained when liver soluble fraction was added to microsomal fraction in the presence of a physiological level of GSH (2 mM), whereas at 20 mM GSH the effect was merely additive. The microsomal component of the liver system was labile, had maximal activity around pH 7.5, and was exceedingly sensitive to NaAsO2 (93% inhibition by 10(-6) M arsenite in the presence of a 20,000-fold excess of GSH). The microsomal activity apparently results from a Se-methyltransferase, possibly a dithiol protein, that methylates hydrogen selenide produced enzymically by the soluble fraction or non-enzymically when a sufficiently high concentration of GSH is used.

NADPH activation of deoxycytidylate kinase in rat liver extract: Involvement of an endogenous disulfide reductase system

Deoxycytidylate kinase (EC 2.7.4.14) in cell-free extract of rat liver, whose activity was highly dependent on the presence of high concentrations of thiol compound during incubation prior to assay, could be activated by substituting 0.1 to 0.2 mM NADPH for the thiol. Partial purification of the extract resulted in a separation of components indicating that the NADPH-dependent activation system was composed of, other than kinase itself, at least two protein factors: one was heat-stable and the other was indistinguishable from NADPH-dependent disulfide reductase [4]. Similarity of this system to thioredoxin-thioredoxin reductase system [7] is noted.