Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.8.1.12 (trypanothione reductase)
355 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Trypanothione reductase was purified to homogeneity from Leishmania donovani promastigotes transfected with the expression plasmid pTEX-LdTR. The physical, spectral and kinetic properties were found to be similar to those obtained from other pathogenic trypanosomatids. The substrates trypanothione disulfide and NADPH exhibit Michaelis-Menten saturation kinetics with Km values of 36 microM and 9 microM, respectively, the former yielding a kcat/Km of 5.0 x 10(6) M-1 s-1. Like other trypanothione reductases, the leishmania enzyme is unable to use glutathione disulfide as substrate. Both trypanothione reductase and the analogous mammalian enzyme, glutathione reductase, are inhibited by trivalent but not pentavalent anti-leishmanial antimonials. Inhibition by trivalent sodium antimonyl gluconate (Triostam) occurs in a time-dependent manner, with the pseudo-first-order rate constants of inhibition being linearly related to drug concentration. Inhibition proceeds until an apparent equilibrium between active enzyme/free drug and inactive enzyme-drug complex is reached. MelT, an adduct of melarsen oxide and dihydrotrypanothione which is a competitive inhibitor of the disulfide binding site of trypanothione reductase, confers protection against Triostam. Prior reduction of the catalytically active disulfide bridge by NADPH is essential for inhibition. Spectral analysis shows that the broad absorbance band centred on 530 nm, characteristic of the charge-transfer complex in the two-electron-reduced EH2 enzyme, is lost upon addition of Triostam. Further spectral changes resemble those associated with reduction of the FAD prosthetic group to FADH2. Inhibition by Triostam is readily reversed by dilution or addition of the dithiols 2,3-dimercaptopropanol, 2,3-dimercaptosuccinate or dithiothreitol, but not dihydrotrypanothione, suggesting that this trypanosomatid-unique metabolite is unlikely to protect the enzyme from inhibition in whole cells. A mechanism consistent with these observations is proposed.
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PMID:Trypanothione reductase from Leishmania donovani. Purification, characterisation and inhibition by trivalent antimonials. 760 16

Multiple sequence alignments including the enterococcal NADH peroxidase and NADH oxidase indicate that residues Ser38 and Cys42 align with the two cysteines of the redox-active disulfides found in glutathione reductase (GR), lipoamide dehydrogenase, mercuric reductase, and trypanothione reductase. In order to evaluate those structural determinants involved in the selection of the cysteine-sulfenic acid (Cys-SOH) redox centers found in the two peroxide reductases and the redox-active disulfides present in the GR class of disulfide reductases, NADH peroxidase residues Ser38, Phe39, Leu40, and Ser41 have been individually replaced with Cys. Both the F39C and L40C mutant peroxidases yield active-site disulfides involving the new Cys and the native Cys42; formation of the Cys39-Cys42 disulfide, however, precludes binding of the FAD coenzyme. In contrast, the L40C mutant contains tightly-bound FAD and has been analyzed by both kinetic and spectroscopic approaches. In addition, the L40C and S41C mutant structures have been determined at 2.1 and 2.0 A resolution, respectively, by X-ray crystallography. Formation of the Cys40-Cys42 disulfide bond requires a movement of Cys42-SG to a new position 5.9 A from the flavin-C(4a) position; this is consistent with the inability of the new disulfide to function as a redox center in concert with the flavin. Stereochemical constraints prohibit formation of the Cys41-Cys42 disulfide in the latter mutant.
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PMID:An L40C mutation converts the cysteine-sulfenic acid redox center in enterococcal NADH peroxidase to a disulfide. 771 Oct 38

Nitrofurans with aromatic and heterocyclic substituents inhibit Trypanosoma congolense trypanothione reductase (TR) and yeast glutathione reductase (GR), acting as uncompetitive inhibitors vs. NADPH and noncompetitive or uncompetitive inhibitors vs. disulfide substrate. Many of these compounds inhibited trypanothione reductase more efficiently than glutathione reductase. Chinifur (2-[5'-nitro(furo-2'-yl)-ethene-1-yl]-4(N,N-diethylamino)-1-methyl-but-1 -yl - aminocarbonyl-4-quinoline) was the most selective inhibitor of, and free radical-generating substrate for, trypanothione reductase (Ki = 4.5 microns, TN = 3 s-1, TN/Km = 3.2 x 10(4) M-1 s-1), only weakly inhibiting glutathione reductase (Ki = 100 microns). These findings point to the importance of hydrophobic interactions in the design of redox active heteroaromatic compounds acting as selective inhibitors of, and "subversive substrates" for, trypanothione reductase.
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PMID:Chinifur, a selective inhibitor and "subversive substrate" for Trypanosoma congolense trypanothione reductase. 794 63

The three-dimensional structure of trypanothione reductase (TR) (EC 1.6.4.8) from Trypanosoma cruzi has been solved at 0.33 nm resolution by molecular replacement using the structure of C. fasciculata TR as a starting model. Elucidation of the T. cruzi TR structure represents the first step in the rational design of a drug against Chagas' disease. The structure of T. cruzi TR is compared with those of C. fasciculata TR as well as human and E. coli glutathione reductase (GR). In the FAD-binding domain, TR has two insertions, each about 10 residues long, which do not occur in GR. The first one is a rigid loop stabilizing the position of helix 91-117 which is responsible for the wider active site of TR as compared to GR. The second insertion does not occur where it is predicted by sequence alignment; rather the residues extend three strands of the 4-stranded beta-sheet by one or two residues each. This increases the number of hydrogen bonds within the sheet structure. The structure of the NADPH.TR complex has been solved at 0.33 nm resolution. The nicotinamide ring is sandwiched between the flavin ring and the side chain of Phe-198 which undergoes the same conformational change upon coenzyme binding as Tyr-197 in GR. In addition to Arg-222 and Arg-228, which are conserved in TR and GR, Tyr-221--the last residue of the second beta-sheet strand of the beta alpha beta dinucleotide binding fold--is in hydrogen bonding distance to the 2' phosphate group of NADPH.
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PMID:The structure of Trypanosoma cruzi trypanothione reductase in the oxidized and NADPH reduced state. 815 65

A cDNA encoding soybean (Glycine max [L.] Merr) ferric leghemoglobin reductase (FLbR), an enzyme that is postulated to play an important role in maintaining leghemoglobin in its functional ferrous state, has been cloned and characterized. A group of highly degenerate oligonucleotides deduced from the N-terminal amino acid sequence of FLbR was used to prime the polymerase chain reaction (PCR) on soybean nodule mRNA and cDNA. A full-length clone of FLbR cDNA was isolated by screening a lambda gt11 soybean nodule cDNA library using the specific PCR-amplified FLbR cDNA fragment as a probe. The cDNA contained about 1.8 kb and had a coding sequence for 523 amino acids with a predicted molecular mass of 55,729 D, which included a putative 30-residue signal peptide and a 493-residue mature protein. Computer-aided analysis of the deduced FLbR amino acid sequence showed considerable homology (varied from 20-50% with enzymes and species) to dihydrolipoamide dehydrogenase (EC 1.8.1.4), glutathione reductase (EC 1.6.4.2), mercuric reductase (EC 1.16.1.1), and trypanothione reductase (EC 1.6.4.8) in a superfamily of pyridine nucleotide-disulfide oxidoreductases from various organisms. Northern blot analysis using FLbR cDNA as a probe showed that the FLbR gene was expressed in soybean nodules, leaves, roots, and stems, with a greater level of expression in nodules and leaves than in roots and stems. Southern blot analysis of the genomic DNA showed the presence of two homologous FLbR genes in the soybean genome.
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PMID:Cloning and sequence analysis of a cDNA encoding ferric leghemoglobin reductase from soybean nodules. 815 85

The dithiol trypanothione, novel to trypanosomatids and analogous to glutathione in mammalian systems, has been shown to interact with anti-trypanocidal trivalent arsenical drugs forming a stable adduct, MelT. This adduct is a competitive inhibitor of the flavoprotein trypanothione reductase, responsible for maintaining intracellular trypanothione in the reduced form. Since trypanothione reductase and the analogous glutathione reductase both contain catalytically active sulphydryl groups we have examined the ability of several arsenicals to differentially inhibit these enzymes. Melarsen oxide [p-(4,6-diamino-s-triazin-2-yl)aminophenylarsenoxide] potently inhibits both enzymes in two stages, the first being essentially complete within 1 min, the second being time dependent, exhibiting saturable pseudo-first-order kinetics with kinact of 14.3 x 10(-4) s-1 and 1.06 x 10(-4) s-1 and Ki of 17.2 microM and 9.6 microM for trypanothione reductase and glutathione reductase, respectively. Inhibition requires prior reduction of the enzyme by NADPH and can be reversed by excess dithiols or prevented by MelT in the case of trypanothione reductase. In both cases a time-dependent loss of the characteristic charge-transfer absorbance band at 530 nm is observed upon addition of arsenical to pre-reduced enzyme, which with excess NADPH leads to a spectrum resembling the EH4 form and is accompanied by an increased ability to reduce molecular oxygen. A model for inhibition is proposed where, first, free arsenical and previously reduced enzyme immediately establish an equilibrium with an inactive monothioarsane enzyme-inhibitor complex involving the interchange cysteine distal to the FAD; second, a subsequent rearrangement about the sulphur-arsenic bond leads to the binding of the arsenical to the charge-transfer cysteine, proximal to the FAD, forming a more stable dithioarsane complex. Molecular modelling suggests that the differences in kinetic behaviour of the two enzymes can be attributed to structural features of their respective disulphide-binding sites. Incubation of reduced trypanothione reductase with excess dihydrotrypanothione and melarsen oxide prevents direct inhibition of the enzyme, suggesting that dihydrotrypanothione acts as a protectant in vivo, preventing the direct modification of trypanothione reductase by sequestering the arsenical as MelT.
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PMID:Mechanism of inhibition of trypanothione reductase and glutathione reductase by trivalent organic arsenicals. 816 18

The irradiation of Trypanosoma congolense trypanothione reductase (TR), human erythrocyte (HGR) and yeast glutathione reductase (YGR) with visible light in the presence of Al-phthalocyanine tetrasulfonate (A1PcS4) or hematoporphyrine (Hp) caused a time-dependent inactivation of these enzymes. TR was inactivated more rapidly than either HGR or YGR. Half-maximal rates of inactivation were determined in the presence of 100 microM Hp and 1.4-17 microM AlPcS4. The photosensitized irradiation modified the disulfide substrate-binding sites of these enzymes, most likely the conserved catalytic histidine residue. In the dark, AlPcS4 acted as a reversible inhibitor competitive with the disulfide substrate of TR and HGR. These findings suggest the possible use of photosensitized irradiation for preventing the transmission of trypanosomiasis by blood transfusion.
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PMID:Photoinactivation of trypanothione reductase and glutathione reductase by Al-phthalocyanine tetrasulfonate and hematoporphyrin. 856 7

The three-dimensional structure of the complex between Trypanosoma cruzi trypanothione reductase (TR) (EC 1.6.4.8) and the antiparasitic drug mepacrine (quinacrine) has been solved at 2.9 angstoms resolution. Mepacrine is a competitive inhibitor of TR but does not affect human glutathione reductase (GR), a closely related host enzyme. Of particular importance for inhibitor binding are four amino acid residues in the disulfide substrate-binding site of TR that are not conserved in human GR, namely, Glu-18 (Ala-34 in GR), Trp-21 (Arg-37), Ser-109 (Ile-113), and Met-113 (Asn-117). The acridine ring of mepacrine is fixed at the active site close to the hydrophobic wall formed by Trp-21 and Met-113. Specific pairwise interactions between functional groups of the drug and amino acid side chains include the ring nitrogen and Met-113, the chlorine atom and Trp-21, and the oxymethyl group and Ser-109. The alkylamino chain of mepacrine points into the inner region of the active site and is held in position by a solvent-mediated hydrogen bond to Glu-18. The structure of the complex shows for the first time the atomic interactions between TR and an inhibitory ligand. This is a crucial step towards the rational design of inhibitors that might be suited as drugs against Chagas' disease.
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PMID:Crystal structure of the Trypanosoma cruzi trypanothione reductase.mepacrine complex. 862 34

Benson et al. (Biochem. J. 1992, 286, 9) reported three novel competitive inhibitors of trypanothione reductase (TR), which were selected to complement a hydrophobic region identified on the TR structure which was not present on human glutathione reductase (hGR). Benson et al. also noted that chlorpromazine, a tricyclic antidepressant known to have trypanocidal activity, was an inhibitor of TR. Here we show that chlorpromazine is a competitive inhibitor of TRs from Crithidia fasciculata (Ki = 14 microM) and Trypanosoma cruzi (Ki = 10 microM), but the drug binds > 50-fold more weakly (Ki = 762 microM) to hGR. Analogues of chlorpromazine differing in the length of the side chain carrying the positively charged R-group are also selective TR inhibitors whereas, a tricyclic structure carrying a negatively charged side chain is a competitive inhibitor with selectivity for hGR (K(hGR)i = 165 microM vs. K(TR)i = 1400 microM). This finding suggests that simple charge characteristics, rather than differences in hydrophobicity, may account for a significant portion of the selectivity of this series of inhibitors for these two enzymes. Electrostatic analysis of the structures of TR and hGR thus provides a rationale for these results, and offers a new principle for inhibitor design. The principle gains further support from the observation that all known tricyclic competitive inhibitors of TR are positively charged. In order to investigate the in vivo relevance of our findings we have examined the effect of chlorpromazine and its negatively charged analogue on the growth of C. fasciculata parasites. Consistent with our kinetics, chlorpromazine (50 microM) inhibited the growth of parasites by 50%, while no measurable decrease in parasite growth rate was noted in the presence of the negatively charged inhibitor (400 microM). Furthermore, the highly similar inhibitory profiles of C. fasciculata TR and T. cruzi TR suggest that drug-design studies using the structurally better-studied C. fasciculata TR are also relevant to the human pathogen T. cruzi.
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PMID:Charge is the major discriminating factor for glutathione reductase versus trypanothione reductase inhibitors. 887 46

Oocysts of Cryptosporidium parvum showed relatively low levels of SOD activity. The SOD which had a pI of 4.8 and an approximate molecular weight of 35 kDa appeared to be iron dependent. Catalase, glutathione transferase, glutathione reductase and glutathione peroxidase activity could not be detected, nor could trypanothione reductase. No NADH or NADPH oxidase activity could be detected, nor could peroxidase activity be demonstrated using o-dianisidine, guaiacol, NADPH or NADH as co-substrates. However, an NADPH-dependent H2O2 scavenging system was detected in the insoluble fraction.
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PMID:Anti-oxidant enzymes in Cryptosporidium parvum oocysts. 901 Oct 70


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