Sllmmal~Tuberculosis remains one of the major infectious causes of morbidity and mortality in the world, yet the mechanisms by which macrophages defend against Mycobacterium tuberculosis have remained obscure. Results from this study show that murine macrophages, activated by interferon % and lipopolysaccharide or tumor necrosis factor or, both growth inhibit and kill M. tuberculosis. This antimycobacterial effect, demonstrable both in murine macrophage cell lines and in peritoneal macrophages of BALB/c mice, is independent of the macrophage capacity to generate reactive oxygen intermediates (ROI). Both the ROI-deficient murine macrophage cell line Dg, and its ROI-generating, parental line J774.16, expressed comparable antimycobacterial activity upon activation. In addition, the oxygen radical scavengers superoxide dismutase (SOD), catalase, mannitol, and diazabicyclooctane had no effect on the antimycobacterial activity of macrophages. These findings, together with the results showing the relative resistance of M. tuberculosis to enzymaticaUy generated H202, suggest that ROI are unlikely to be significantly involved in killing M. tuberculosis. In contrast, the antimycobacterial activity of these macrophages strongly correlates with the induction of the t-arginine-dependent generation of reactive nitrogen intermediates (RNI). The effector molecule(s) that could participate in mediating this antimycobacterial function are toxic RNI, including NO, NO2, and HNO2, as demonstrated by the micobacteriocidal effect of acidified NO2. The oxygen radical scavenger SOD adventitiously perturbs RNI production, and cannot be used to discriminate between cytocidal mechanisms involving ROI and RNI. Overall, our results provide support for the view that the r-arginine-dependent production of RNI is the principal effector mechanism in activated murine macrophages responsible for killing and growth inhibiting virulent M. tuberculosis.
Resonance Raman spectra of native, overexpressed M. tuberculosis catalase-peroxidase (KatG), the enzyme responsible for activation of the antituberculosis antibiotic isoniazid (isonicotinic acid hydrazide), have confirmed that the heme iron in the resting (ferric) enzyme is high-spin five-coordinate. Difference Raman spectra did not reveal a change in coordination number upon binding of isoniazid to KatG. Stopped-flow spectrophotometric studies of the reaction of KatG with stoichiometric equivalents or small excesses of hydrogen peroxide revealed only the optical spectrum of the ferric enzyme with no hypervalent iron intermediates detected. Large excesses of hydrogen peroxide generated oxyferrous KatG, which was unstable and rapidly decayed to the ferric enzyme. Formation of a pseudo-stable intermediate sharing optical characteristics with the porphyrin pi-cation radical-ferryl iron species (Compound I) of horseradish peroxidase was observed upon reaction of KatG with excess 3-chloroperoxybenzoic acid, peroxyacetic acid, or tert-butylhydroperoxide (apparent second-order rate constants of 3.1 x 10(4), 1.2 x 10(4), and 25 M(-1) s(-1), respectively). Identification of the intermediate as KatG Compound I was confirmed using low-temperature electron paramagnetic resonance spectroscopy. Isoniazid, as well as ascorbate and potassium ferrocyanide, reduced KatG Compound I to the ferric enzyme without detectable formation of Compound II in stopped-flow measurements. This result differed from the reaction of horseradish peroxidase Compound I with isoniazid, during which Compound II was stably generated. These results demonstrate important mechanistic differences between a bacterial catalase-peroxidase and the homologous plant peroxidases and yeast cytochrome c peroxidase, in its reactions with peroxides as well as substrates.
Inhibition of the enzyme Mycobacterium tuberculosis InhA (enoyl-acyl carrier protein reductase) due to formation of an isonicotinoyl-NAD adduct (IN-NAD) from isoniazid (INH) and nicotinamide adenine dinucleotide cofactor is considered central to the mode of action of INH, a first-line treatment for tuberculosis infection. INH action against mycobacteria requires catalase-peroxidase (KatG) function, and IN-NAD adduct formation is catalyzed in vitro by M. tuberculosis KatG under a variety of conditions, yet a physiologically relevant approach to the process has not emerged that allows scrutiny of the mechanism and the origins of INH resistance in the most prevalent drug-resistant strain bearing KatG[S315T]. In this report, we describe how hydrogen peroxide, delivered at very low concentrations to ferric KatG, leads to efficient inhibition of InhA due to formation of the IN-NAD adduct. The rate of adduct formation mediated by wild-type KatG was about 20-fold greater than by the isoniazid-resistant KatG[S315T] mutant under optimal conditions (H2O2 supplied along with NAD+ and INH). Slow adduct formation also occurs starting with NADH and INH, in the presence of KatG even in the absence of added peroxide, due to endogenous peroxide. The poor efficiency of the KatG[S315T] mutant can be enhanced merely by increasing the concentration of INH, consistent with this enzyme's reduced affinity for INH binding to the resting enzyme and the catalytically competent enzyme intermediate (Compound I). Origins of drug resistance in the KatG[S315T] mutant enzyme are analyzed at the structural level through examination of the three-dimensional X-ray crystal structure of the mutant enzyme.
The unique antitubercular activity of isoniazid requires that the drug be oxidized by the katG-encoded mycobacterial catalase-peroxidase to an activated drug form. In order to quantitatively assess the catalytic capabilities of the enzyme, the native catalase-peroxidase from Mycobacterium smegmatis was purified over 200-fold to homogeneity. The enzyme was shown to exhibit both catalase and peroxidase activities, and in the presence of either hydrogen peroxide or t-butyl peroxide, was found to catalyze the oxidation of the reduced pyridine nucleotides, NADH and NADPH, as well as artificial peroxidase substrates, at rates between 2.7 and 20 s ؊1 . The homogeneous enzyme exhibited a visible absorbance spectrum typical of ferric heme-containing catalase-peroxidases, with a Soret maximum at 406 nm. Low temperature (10 K) electron paramagnetic resonance spectra in the presence of ethylene glycol revealed a high spin Fe(III) signal with g values of 5.9 and 5.6. The enzyme was very slowly (t1 ⁄2 ؍ ϳ 20 min) reduced by dithionite, and the reduced form showed typical spectral changes when either KCN or CO were subsequently added. The M. smegmatis catalase-peroxidase was found to contain 2 heme molecules per tetramer, which were identified as iron protoporphyrin IX by the pyridine hemochromogen assay. The peroxidatic activity was inhibited by KCN, NaN 3 , isoniazid (isonicotinic acid hydrazide), and its isomer, nicotinic acid hydrazide, but not by 3-amino-1,2,4-triazole. The role of mycobacterial catalase-peroxidases in the oxidative activation of the antitubercular prodrug isoniazid is discussed.Metabolism in an oxygen-containing environment results in the generation of reactive oxygen species such as superoxide, hydroxyl radical, and hydrogen peroxide. Organisms have evolved sophisticated and efficient enzyme systems to neutralize these potentially injurious reactive oxygen species, including catalases, superoxide dismutases, and peroxidases. Enzymes that exhibit either catalatic or peroxidatic activity are common among animals, plants, and microorganisms, while bacteria and some fungi have been found to contain hydroperoxidases that exhibit both catalase and peroxidase activities. These catalase-peroxidases have been purified from Escherichia coli B (1), Klebsiella pneumoniae (2), Halobacterium halobium (3), Septoria tritici (4), and a Bacillus strain YN-2000 (5), and exhibit visible absorbance spectra typical of a ferric high spin hemoprotein. The pyridine ferrohemochromes of these enzymes show an absorption spectrum typical of ferric protoheme IX (1-5). A characteristic feature of the bacterial catalase-peroxidases is their low heme content, with only 2 molecules of heme per enzyme tetramer. The activity of bacterial catalase-peroxidases is inhibited by azide and cyanide, but not by the catalase-specific inhibitor, 3-amino-1,2,4-triazole.The enzymological characterization of mycobacterial catalase-peroxidases is relevant for a number of reasons. Human mycobacterial pathogens, such as Mycobacterium tuberculosis, Myc...
Stable films of dimyristoylphosphatidylcholine and M. tuberculosis catalase-peroxidase (KatG), several peroxidases, myoglobin, and catalase showed reversible FeIII/FeII voltammetry on pyrolytic graphite electrodes and catalytic current for hydrogen peroxide and oxygen. Amperometric responses for these films to H2O2 at 0 V are likely to contain significant contributions from catalytic reduction of oxygen produced during the catalytic cycles. Relative apparent turnover rates at pH 6 based on steady-state currents at 0 V versus SCE in the presence of H2O2 were in the order horseradish peroxidase > cytochrome c peroxidase (CcP) > soybean peroxidase > myoglobin > KatG > catalase. Lower currents for the very efficient peroxide scavengers KatG and catalase may be related to the instability of their compounds I in the presence of H2O2. KatG catalyzed the electrochemical reduction of oxygen more efficiently than catalase and CcP but less efficiently than the other peroxidases. DMPC films incorporating glucose oxidase and peroxidases gave good analytical responses to glucose, demonstrating the feasibility of dual enzyme-lipid films for biosensor fabrication.
Catalase-peroxidase (KatG) from Mycobacterium tuberculosis is responsible for the activation of the antitubercular drug isonicotinic acid hydrazide (INH) and is important for survival of M. tuberculosis in macrophages. Characterization of the structure and catalytic mechanism of KatG is being pursued to provide insights into drug (INH) resistance in M. tuberculosis. Site-directed mutagenesis was used to prepare the INH-resistant mutant KatG[S315T], and the overexpressed enzyme was characterized and compared with wild-type KatG. KatG[S315T] exhibits a reduced tendency to form sixcoordinate heme, because of coordination of water to iron during purification and storage, and also forms a highly unstable Compound III (oxyferrous enzyme). Catalase activity and peroxidase activity measured using t-butylhydroperoxide and o-dianisidine were moderately reduced in the mutant compared with wild-type KatG. Stopped-flow spectrophotometric experiments revealed a rate of Compound I formation similar to wildtype KatG using peroxyacetic acid to initiate the catalytic cycle, but no Compound I was detected when bulkier peroxides (chloroperoxybenzoic acid, t-butylhydroperoxide) were used. The affinity of resting (ferric) KatG[S315T] for INH, measured using isothermal titration calorimetry, was greatly reduced compared with wild-type KatG, as were rates of reaction of Compound I with the drug. These observations reveal that although KatG[S315T] maintains reasonably good steady state catalytic rates, poor binding of the drug to the enzyme limits drug activation and brings about INH resistance.
Pyruvate formate-lyase (PFL) catalyzes the reversible conversion of CoA and pyruvate into acetyl-CoA and formate. Active enzyme contains a glycyl radical whose alpha-hydrogen undergoes rapid exchange with solvent (t1/2 approximately 5 min at 0 degree C). We have investigated this exchange using site-directed mutagenesis and mechanism-based inactivation. Mutation of the active-site cysteine 419 into a serine, which renders the enzyme catalytically inactive, abolishes alpha-hydrogen exchange in the radical. This suggests that the exchange process is not an intrinsic property of the glycyl radical but is a consequence of its interaction with cysteine 419. This residue is also demonstrated to be involved in the transfer of the radical to acetylphosphinate, a mechanism-based inactivator of the enzyme. In contrast, mutation of the other essential cysteine 418 to a serine has no effect on the hydrogen exchange or the transfer of the radical to acetylphosphinate. A mechanism for the hydrogen exchange catalyzed by cysteine 419 consistent with a redox role for this residue in the normal catalytic reaction is proposed.
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