Metabolic enzymes of mycobacteria linked to antioxidant defense by a thioredoxin-like protein.

Mycobacterium tuberculosis (Mtb) mounts a stubborn defense against oxidative and nitrosative components of the immune response. Dihydrolipoamide dehydrogenase (Lpd) and dihydrolipoamide succinyltransferase (SucB) are components of alpha-ketoacid dehydrogenase complexes that are central to intermediary metabolism. We find that Lpd and SucB support Mtb's antioxidant defense. The peroxiredoxin alkyl hydroperoxide reductase (AhpC) is linked to Lpd and SucB by an adaptor protein, AhpD. The 2.0 angstrom AhpD crystal structure reveals a thioredoxin-like active site that is responsive to lipoamide. We propose that Lpd, SucB (the only lipoyl protein detected in Mtb), AhpD, and AhpC together constitute a nicotinamide adenine dinucleotide (reduced)-dependent peroxidase and peroxynitrite reductase. AhpD thus represents a class of thioredoxin-like molecules that enables an antioxidant defense.

Peroxynitrite reductase activity of bacterial peroxiredoxins.

Nitric oxide (NO) is present in soil and air, and is produced by bacteria, animals and plants. Superoxide (O2-) arises in all organisms inhabiting aerobic environments. Thus, many organisms are likely to encounter peroxynitrite (OONO-), a product of NO and O2- that forms at near diffusion-limited rates, and rapidly decomposes upon protonation through isomerization to nitrate (NO3-; ref. 1) while generating hydroxyl radical (*OH) and nitrogen dioxide radical (*NO2) (refs 2, 3), both more reactive than peroxynitrite's precursors. The oxidative, inflammatory, mutagenic and cytotoxic potential (ref. 4) of peroxynitrite contrasts with the antioxidant, anti-inflammatory and tissue-protective properties ascribed to NO itself. Thus, the ability of cells to cope with peroxynitrite is central in determining the biological consequences of NO production. We considered whether cells might be equipped with enzymes to detoxify peroxynitrite. Peroxiredoxins have been identified in most genomes sequenced, but their functions are only partly understood. Here we show that the peroxiredoxin alkylhydroperoxide reductase subunit C (AhpC) from Salmonella typhimurium catalytically detoxifies peroxynitrite to nitrite fast enough to forestall the oxidation of bystander molecules such as DNA. Results are similar with peroxiredoxins from Mycobacterium tuberculosis and Helicobacter pylori. Thus, peroxynitrite reductase activity may be widespread among bacterial genera.

Pharmacological modulation of nitric oxide synthesis by mechanism-based inactivators and related inhibitors.

Nitric oxide synthase (NOS) (EC 1.14.13.39) is a homodimeric cytochrome P450 monooxygenase analog that generates nitric oxide (NO) from the amino acid L-arginine. Enzymatically produced NO acts as an intracellular messenger in neuronal networks, blood pressure regulatory mechanisms, and immune responses. Isoform-selective pharmacological modulation of NO synthesis has emerged as a new therapeutic strategy for the treatment of diverse clinical conditions associated with NO overproduction. Mechanism-based inactivators (MBIs) represent a class of NOS mechanistic inhibitors that require catalytic turnover to produce irreversible inactivation of the ability of NOS to generate NO. Diverse isoform-selective NOS MBIs have been characterized with respect to their kinetic parameters and chemical mechanisms of inactivation. In studies with isolated and purified NOS isoforms, MBIs produce irreversible inactivation of NOS enzymatic activities. The inactivation process is associated with covalent modification of the NOS active site and proceeds either through heme destruction, its structural alteration, or covalent modification of the NOS protein chain. The behavior of NOS MBIs in intact cells is different from their behavior observed with the isolated NOS isoforms. In cytokine-induced RAW 264.7 macrophages, treatment with MBIs produces a complete loss of cellular NOS synthetic competence and inducible NOS activity. However, following drug removal, cells can recover at least partially in the absence of protein synthesis. In GH3 cells containing the neuronal NOS isoform, calcium transients are too low and abbreviated to allow significant NOS inactivation; hence, the cellular effects of MBIs on the neuronal isoform are almost completely and immediately reversible.

Studies of neuronal nitric oxide synthase inactivation by diverse suicide inhibitors.

N(G)-Amino-l-arginine, N(5)-(1-iminoethyl)-l-ornithine, N(6)-(1-iminoethyl)-l-lysine, and aminoguanidine were studied for the mechanisms by which they produce suicidal inactivation of the neuronal nitric oxide synthase isoform (nNOS). All of the inactivators that were amino acid structural analogs targeted the heme residue at the nNOS active site and led to its destruction as evidenced by the time- and concentration-dependent loss of the nNOS heme fluorescence, which reflects the disruption of the protoporphyrin-conjugated structure. The loss of heme was exclusively associated with the dimeric population of the nNOS. This inactivator-mediated loss of the nNOS heme never reached more than 60%, suggesting that only half of the dimeric heme is involved in catalytic activation of mechanism-based inactivators studied. Aminoguanidine-induced nNOS inactivation produced covalent modification of the nNOS protein chain with a stoichiometry of 0.8 mol of aminoguanidine per mole of the nNOS monomer. Specific covalent modification by aminoguanidine was exclusively associated with the oxygenase domain of the nNOS. The mechanisms by which N(6)-(1-iminoethyl)-l-lysine and aminoguanidine inactivate the nNOS and iNOS do not differ between the isoforms. The selectivity of these inactivators toward the iNOS isoform is a reflection of their much lower partition ratios, which were determined to be 0.16 +/- 0. 1 for N(6)-(1-iminoethyl)-l-lysine and 12 +/- 1.5 for aminoguanidine in case of the iNOS isoform while the same inactivators produced the partition ratios of 17 +/- 2 and 206 +/- 4, respectively, for the nNOS isoform.

Mechanism of inducible nitric oxide synthase inactivation by aminoguanidine and L-N6-(1-iminoethyl)lysine.

The inducible nitric oxide synthase (iNOS) selective inhibitors aminoguanidine (AG) and N6-(1-iminoethyl)-L-lysine (NIL), under conditions that support catalytic turnover, inactivate the enzyme by altering in different ways the functionality of the active site. NIL inactivation of the iNOS primarily targets the heme residue at the active site, as evidenced by a time- and concentration-dependent loss of heme fluorescence that accompanies the loss of NO-forming activity. The NIL-inactivated iNOS dimers that have lost their heme partially disassemble into monomers with no fluorometrically detectable heme. AG inactivation of the iNOS is not accompanied by heme destruction, as evidenced by retention of heme fluorescence and absorbance after complete loss of NO-forming activity. The AG-inactivated iNOS dimers do not disassemble into monomers as extensively as NIL-inactivated dimers. Incubation of the iNOS with 14C-labeled NIL results in no detectable protein-associated radioactivity in the NIL-inactivated iNOS, suggesting that the primary mechanism of the iNOS inactivation by NIL is heme alteration and loss. In contrast, incubations of iNOS with 14C-labeled AG result in the incorporation of radioactivity into both iNOS protein and low molecular weight structures that migrate by SDS-PAGE similarly to free heme. These observations suggest that AG inactivation proceeds through multiple pathways of covalent modification of the iNOS protein and the heme residue at the active site, but which sustain the integrity of the heme porphyrin ring.

Pharmacological characterization of guanidinoethyldisulphide (GED), a novel inhibitor of nitric oxide synthase with selectivity towards the inducible isoform.

1. Guanidines, amidines, S-alkylisothioureas, and recently, mercaptoalkylguanidines have been described as inhibitors of the generation of nitric oxide (NO) from L-arginine by NO synthases (NOS). We have recently demonstrated that guanidinoethyldisulphide (GED), formed from the dimerisation of mercaptoethylguanidine (MEG), is a novel inhibitor of nitric oxide synthases. Here we describe the pharmacological properties of GED on purified NOS isoforms, various cultured cell types, vascular ring preparations, and in endotoxin shock. 2. GED potently inhibited NOS activity of purified inducible NOS (iNOS), endothelial NOS (ecNOS), and brain NOS (bNOS) enzymes with Ki values of 4.3, 18 and 25 microM, respectively. Thus, GED has a 4 fold selectivity for iNOS over ecNOS at the enzyme level. The inhibitory effect of GED on ecNOS and iNOS was competitive vs. L-arginine and non-competitive vs. tetrahydrobiopterin. 3. Murine J774 macrophages, rat aortic smooth muscle cells, murine lung epithelial cells, and human intestinal DLD-1 cells were stimulated with appropriate mixtures of pro-inflammatory cytokines or bacterial lipopolysaccharide to express iNOS. In these cells, GED potently inhibited nitrite formation (EC50 values: 11, 9, 1 and 30 microM, respectively). This suggests that uptake of GED may be cell type and species-dependent. The inhibitory effect of GED on nitrite production was independent of whether GED was given together with immunostimulation or 6 h afterwards, indicating that GED does not interfere with the process of iNOS induction. 4. GED caused relaxations in the precontracted vascular ring preparations (EC50: 20 microM). Part of this relaxation was endothelium-dependent, but was not blocked by methylene blue (100 microM), an inhibitor of soluble guanylyl cyclase. In precontracted rings, GED enhanced the acetylcholine-induced, endothelium-dependent relaxations at 10 microM and caused a slight inhibition of the relaxations at 100 microM. The vascular studies demonstrate that the inhibitory potency of GED on ecNOS in the ring preparations is considerably lower than its potency against iNOS in the cultured cells. These data suggest that the selectivity of GED towards iNOS may lie, in part, at the enzyme level, as well as differential uptake by cells expressing the various isoforms of NOS. 5. In a rat model of endotoxin shock in vivo, administration of GED, at 3 mg kg-1 bolus followed by 10 mg kg-1 h-1 infusion, starting at 90 min after bacterial lipopolysaccharide (LPS, 15 mg kg-1, i.v.), prevented the delayed fall in mean arterial blood pressure, prevented the development of the vascular hyporeactivity to noradrenaline of the thoracic aorta ex vivo and protected against the impairment of the endothelium-dependent relaxations associated with this model of endotoxaemia. The same bolus and infusion of the inhibitor did not alter blood pressure or ex vivo vascular reactivity in normal animals over 90 min. 6. Administration of GED (10 mg kg-1, i.p.) given at 2 h after LPS (120 mg kg-1, i.p.) and every 6 h thereafter caused a significant improvement in the survival rate in a lethal model of endotoxin shock in mice between 12 and 42 h. 7. In conclusion, we found that GED is a competitive inhibitor of iNOS activity. Its selectivity towards iNOS may lie both at the enzyme level and at the level of cell uptake. GED has beneficial effects in models of endotoxin shock that are driven by iNOS. GED or its derivatives may be useful tools in the experimental therapy of inflammatory conditions associated with NO overproduction due to iNOS expression.