Correlation between isoniazid resistance and superoxide reactivity in mycobacterium tuberculosis KatG.

Isoniazid is an antituberculosis prodrug that requires activation by the catalase-peroxidase (KatG) of Mycobacterium tuberculosis. The activated species, presumed to be an isonicotinoyl radical, couples to NADH forming an isoniazid-NADH adduct that ultimately confers antitubercular activity. We have compared the catalytic properties of three KatGs associated with isoniazid resistance (resistance mutation KatGs, (RM)KatGs: R104L, H108Q, S315T) to wild-type enzyme and two additional lab mutations (wild-type phenotype KatGs, (WTP)KatGs: WT KatG, Y229F, R418L). Neither catalase nor peroxidase activities, nor the presence/absence of the Met-Tyr-Trp cross-link (as probed by LC/MS on tryptic digests of the protein), exhibited any correlation with isoniazid resistance. The yields of isoniazid-NADH adduct formed were determined to be 1-5, 4-12, and 20-70-fold greater for the (WTP)KatGs than the (RM)KatGs for the compound I, II, and III pathways, respectively, strongly suggesting a role for oxyferrous KatG (supported by superoxide consumption measurements) that correlates with drug resistance. Stopped-flow UV-visible spectroscopic studies revealed that all KatGs were capable of forming both compound II and III intermediates. Rates of compound II decay were accelerated 4-12-fold in the presence of isoniazid (vs absence) for the (WTP)KatGs but were unaffected by the drug for the (RM)KatGs. A mechanism for isoniazid resistance which accounts for the observed reactivity for each of the compound I, II, and III intermediates is proposed and suggests that the compound III pathway may be the primary factor in determining overall isoniazid resistance by specific KatG mutants, with secondary contributions arising from the compound I and II pathways.

Requirements for nitric oxide generation from isoniazid activation in vitro and inhibition of mycobacterial respiration in vivo.

Isoniazid (INH), a front-line antituberculosis agent, is activated by mycobacterial catalase-peroxidase KatG, converting INH into bactericidal reactive species. Here we investigated the requirements and the pathway of nitric oxide (NO*) generation during oxidative activation of INH by Mycobacterium tuberculosis KatG in vitro. We also provide in vivo evidence that INH-derived NO* can inhibit key mycobacterial respiratory enzymes, which may contribute to the overall antimycobacterial action of INH.

Nitric oxide generated from isoniazid activation by KatG: source of nitric oxide and activity against Mycobacterium tuberculosis.

Isonicotinic acid hydrazide (INH) is a frontline antituberculosis agent. Once taken up by Mycobacterium tuberculosis, INH requires activation by the catalase-peroxidase KatG, converting INH from its prodrug form into a range of bactericidal reactive species. Here we used 15N-labeled INH together with electron paramagnetic resonance spin trapping techniques to demonstrate that nitric oxide (NO*) is generated from oxidation at the hydrazide nitrogens during the activation of INH by M. tuberculosis KatG. We also observed that a specific scavenger of NO* provided protection against the antimycobacterial activity of INH in bacterial culture. No significant increases in mycobacterial protein nitration were detected, suggesting that NOdot; and not peroxynitrite, a nitrating metabolite of NO*, is involved in antimycobacterial action. In conclusion, INH-derived NO* has biological activity, which directly contributes to the antimycobacterial action of INH.

Purification and characterization of Mycobacterium tuberculosis KatG, KatG(S315T), and Mycobacterium bovis KatG(R463L).

Isoniazid, a first-line antibiotic used for the treatment of tuberculosis, is a prodrug that requires activation by the Mycobacterium tuberculosis enzyme KatG. The KatG(S315T) mutation causes isoniazid resistance while the KatG(R463L) variation is thought to be a polymorphism. Much of the work to date focused on isoniazid activation by KatG has utilized recombinant enzyme overexpressed in Escherichia coli. In this work, native KatG and KatG(S315T) were purified from M. tuberculosis, and KatG(R463L) was purified from Mycobacterium bovis. The native molecular weight, enzymatic activity, optical, resonance Raman, and EPR spectra, K(D) for isoniazid binding, and isoniazid oxidation rates were measured and compared for each native enzyme. Further, the properties of the native enzymes were compared and contrasted with those reported for recombinant KatG, KatG(S315T), and KatG(R463L) in order to assess the ability of the recombinant enzymes to act as good models for the native enzymes.

Electrochemical studies of the mono-Fe, Fe-Zn, and Fe-Fe metalloisoforms of bacteriophage lambda protein phosphatase.

Bacteriophage lambda protein phosphatase (lambdaPP) is a member of a large superfamily of metallophosphoesterases, including serine/threonine protein phosphatases, purple acid phosphatases, 5'-nucleotidase, and DNA repair enzymes such as Mre11. Members of this family share several common characteristics, including a common phosphoesterase motif, secondary structural fold (betaalphabetaalphabeta), and metal ligand environment, and often accommodate a dinuclear metal center. The identity of the active site metals often differs between family members. Despite the extensive spectroscopic studies of several family members, only the standard redox potential of porcine purple acid phosphate (PAP) has been measured. In this report, we investigate the redox properties of another member of this protein family. The standard redox potentials of the mono-Fe, Fe-Zn, and Fe-Fe metalloisoforms of lambdaPP were determined from anaerobic redox titration experiments. Two different S = 5/2, mono-Fe3+ lambdaPP species were identified: the first with an E/D approximately 0.17, g = 8.9 and 4.8, and an Eo' approximately +130 mV; the second with E/D approximately 0.05, g = 6.7, 5.9, and 4.4, and an Eo' approximately +120 mV. The first and second mono-Fe3+ species are thought to represent Fe present in the M2 and M1 sites, respectively. The addition of Zn2+ to mono-Fe3+ lambdaPP results in a decrease in both mono-Fe3+ species and the appearance of a new S = 5/2, Fe(3+)-Zn2+ species (E/D approximately 0.02, g = 5.9, and an Eo' > +175 mV). The Fe-Fe lambdaPP titration revealed an S = 1/2, Fe(3+)-Fe2+ (g < 2) species with an Eo' > +128 mV. These results suggest that the active site of lambdaPP supports a high oxidation potential for both metal sites and may indicate an equally oxidizing active site for other member metallophosphoesterases.

The Treponema pallidum tro operon encodes a multiple metal transporter, a zinc-dependent transcriptional repressor, and a semi-autonomously expressed phosphoglycerate mutase.

The Treponema pallidum tro operon encodes an ABC transporter (TroABCD), a transcriptional repressor (TroR), and the essential glycolytic enzyme phosphoglycerate mutase (Gpm). The apparently discordant observations that the solute binding protein (TroA) binds Zn2+, whereas DNA binding by TroR in vitro is Mn2+-dependent, have generated uncertainty regarding the identities of the ligand(s) and co-repressor(s) of the permease. Moreover, this operonic structure suggests that Gpm expression, and hence glycolysis, the sole source of ATP for the bacterium, would be suspended during TroR-mediated repression. To resolve these discrepancies, we devised an experimental strategy permitting a more direct assessment of Tro operon function and regulation. We report that (i) apo-TroA has identical affinities for Zn2+ and Mn2+; (ii) the Tro transporter expressed in Escherichia coli imports Zn2+, Mn2+, and possibly iron; (iii) TroR represses transporter expression in E. coli at significantly lower concentrations of Zn2+ than of Mn2+; and (iv) TroR-mediated repression causes a disproportionately greater down-regulation of the transporter genes than of gpm. The much higher concentrations of Zn2+ than of Mn2+ in human body fluids suggests that Zn2+ is both the primary substrate and co-repressor of the permease in vivo. Our data also indicate that Gpm expression and, therefore, glycolysis would not be abrogated when T. pallidum encounters high Zn2+ levels.

Formation of a stable cyano-bridged dinuclear iron cluster following oxidation of the superoxide reductases from Treponema pallidum and Desulfovibrio vulgaris with K(3)Fe(CN)(6).

Superoxide reductases catalyze the monovalent reduction of superoxide anion to hydrogen peroxide. Spectroscopic evidence for the formation of a dinuclear cyano-bridged adduct after K(3)Fe(CN)(6) oxidation of the superoxide reductases neelaredoxin from Treponema pallidum and desulfoferrodoxin from Desulfovibrio vulgaris was reported. Oxidation with K(3)Fe(CN)(6) reveals a band in the near-IR with lambda(max) at 1020 nm, coupled with an increase of the iron content by almost 2-fold. Fourier transform infrared spectroscopy provided additional evidence with CN-stretching vibrations at 2095, 2025-2030, and 2047 cm(-)(1), assigned to a ferrocyanide adduct of the enzyme. Interestingly, the low-temperature electronic paramagnetic resonance (EPR) spectra of oxidized TpNlr reveal at least three different species indicating structural heterogeneity in the coordination environment of the active site Fe ion. Given the likely 6-coordinate geometry of the active site Fe(3+) ion in the ferrocyanide adduct, we propose that the rhombic EPR species can serve as a model of a hexacoordinate form of the active site.

Mn2+ is a native metal ion activator for bacteriophage lambda protein phosphatase.

Bacteriophage lambda protein phosphatase (lambdaPP) is a member of a large family of metal-containing phosphoesterases, including purple acid phosphatase, protein serine/threonine phosphatases, 5'-nucleotidase, and DNA repair enzymes such as Mre11. lambdaPP can be activated several-fold by various divalent metal ions, with Mn(2+) and Ni(2+) providing the most significant activation. Despite the extensive characterization of purified lambdaPP in vitro, little is known about the identity and stoichiometry of metal ions used by lambdaPP in vivo. In this report, we describe the use of metal analysis, activity measurements, and whole cell EPR spectroscopy to investigate in vivo metal binding and activation of lambdaPP. Escherichia coli cells overexpressing lambdaPP show a 22.5-fold increase in intracellular Mn concentration and less dramatic changes in the intracellular concentration of other biologically relevant metal ions compared to control cells that do not express lambdaPP. Phosphatase activity assessed using para-nitrophenylphosphate as substrate is increased 850-fold in cells overexpressing lambdaPP, indicating the presence of metal-activated enzyme in cell lysate. EPR spectra of intact cells overexpressing lambdaPP exhibit resonances previously attributed to mononuclear Mn(2+) and dinuclear [(Mn(2+))(2)] species bound to lambdaPP. Spin quantitation of EPR spectra of intact E. coli cells overexpressing lambdaPP indicates the presence of approximately 40 microM mononuclear Mn(2+)-lambdaPP and 60 microM [(Mn(2+))(2)]-lambdaPP. The data suggest that overexpression of lambdaPP results in a mixture of apo-, mononuclear-Mn(2+), and dinuclear-[(Mn(2+))(2)] metalloisoforms and that Mn(2+) is a physiologically relevant activating metal ion in E. coli.

Zinc-substituted Desulfovibrio gigas desulforedoxins: resolving subunit degeneracy with nonsymmetric pseudocontact shifts.

Desulfovibrio gigas desulforedoxin (Dx) consists of two identical peptides, each containing one [Fe-4S] center per monomer. Variants with different iron and zinc metal compositions arise when desulforedoxin is produced recombinantly from Escherichia coli. The three forms of the protein, the two homodimers [Fe(III)/Fe(III)]Dx and [Zn(II)/Zn(II)]Dx, and the heterodimer [Fe(III)/Zn(II)]Dx, can be separated by ion exchange chromatography on the basis of their charge differences. Once separated, the desulforedoxins containing iron can be reduced with added dithionite. For NMR studies, different protein samples were prepared labeled with (15)N or (15)N + (13)C. Spectral assignments were determined for [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx from 3D (15)N TOCSY-HSQC and NOESY-HSQC data, and compared with those reported previously for [Zn(II)/Zn(II)]Dx. Assignments for the (13)C(alpha) shifts were obtained from an HNCA experiment. Comparison of (1)H-(15)N HSQC spectra of [Zn(II)/Zn(II)]Dx, [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx revealed that the pseudocontact shifts in [Fe(II)/Zn(II)]Dx can be decomposed into inter- and intramonomer components, which, when summed, accurately predict the observed pseudocontact shifts observed for [Fe(II)/Fe(II)]Dx. The degree of linearity observed in the pseudocontact shifts for residues >/=8.5 A from the metal center indicates that the replacement of Fe(II) by Zn(II) produces little or no change in the structure of Dx. The results suggest a general strategy for the analysis of NMR spectra of homo-oligomeric proteins in which a paramagnetic center introduced into a single subunit is used to break the magnetic symmetry and make it possible to obtain distance constraints (both pseudocontact and NOE) between subunits.

Is calcineurin a peroxide-specific sensor in T-lymphocytes?

Calcineurin activity in T-lymphocytes has been shown to be sensitive to H(2)O(2). In this report, we investigate the effects of H(2)O(2)and other physiological oxidants on calcineurin activity both in vivo and in vitro. Intracellular calcineurin activity, as determined by NF-AT phosphorylation state and activity, is inhibited by H(2)O(2) but is insensitive to superoxide and NaOCl. Similarly, treatment of T-lymphocytes with NaOCl and paraquat does not drastically alter the activity of calcineurin in crude cell lysate, while H(2)O(2) causes significant inhibition. Sensitivity to H(2)O(2) and NaOCl in vivo correlates with the half-life of each species in cell medium. The intracellular redox potential is unaffected by H(2)O(2) (100 micro M) or NaOCl (600 micro M), indicating that H(2)O(2) inhibits calcineurin via a direct mechanism that does not involve a change in the cytosolic redox potential. In contrast, calcineurin activity in cell lysate is inhibited by all three oxidants. H(2)O(2) inactivation of calcineurin is rapid, with inactivation occurring in

Oxidative stress increases susceptibility of Mycobacterium tuberculosis to isoniazid.

Isoniazid is a first-line antibiotic used in the treatment of infections caused by Mycobacterium tuberculosis. Isoniazid is a prodrug requiring oxidative activation by the catalase-peroxidase hemoprotein, KatG. Resistance to isoniazid can be obtained by point mutations in the katG gene, with one of the most common being a threonine-for-serine substitution at position 315 (S315T). The S315T mutation is found in more than 50% of isoniazid-resistant clinical isolates and results in an approximately 200-fold increase in the MIC of isoniazid compared to that for M. tuberculosis H37Rv. In the present study we investigated the hypothesis that superoxide plays a role in KatG-mediated isoniazid activation. Plumbagin and clofazimine, compounds capable of generating superoxide anion, resulted in a lower MIC of isoniazid for M. tuberculosis H37Rv and a strain carrying the S315T mutation. These agents did not cause as great of an increase in isoniazid susceptibility in the mutant strain when the susceptibilities were assessed by using the inhibitory concentration that causes a 50% decrease in growth. These results provide evidence that superoxide can play a role in isoniazid activation. Since clofazimine alone has antitubercular activity, the observation of synergism between clofazimine and isoniazid raises the interesting possibility of using both drugs in combination to treat M. tuberculosis infections.

What is the ultimate fate of superoxide anion in vivo?

For three decades, oxidative stress and the role of reactive oxygen species in biology have been extensively studied. Recently, a new interest in these areas has emerged with the discovery of superoxide reductases, a family of familiar bacterial metalloenzymes whose heretofore unknown function has now been apparently revealed. In a series of experiments utilizing genetic, molecular biological, and biochemical methods, these enzymes have been shown to be physiologically competent at removing superoxide. The role of these enzymes and their biological relationship to the well-known superoxide dismutases is discussed.

Superoxide reductase activities of neelaredoxin and desulfoferrodoxin metalloproteins.

Superoxide reductases have now been well characterized from several organisms. Unique biochemical features include the ability of the reduced enzyme to react with O2- but not dioxygen (reduced SORs are stable in an aerobic atmosphere for hours). Future biochemical assays that measure the reaction of SOR with O2- should take into account the difficulties of assaying O2- directly and the myriad of redox reactions that can take place between components in the assay, for example, direct electron transfer between cytochrome c and Dfx. Future prospects include further delineation of the reaction mechanisms, characterization of the putative (hydro)peroxo intermediate, and studies that uncover the components between reduced pyridine nucleotides and SOR in the metabolic pathway responsible for O2- detoxification.

Inhibition of bacteriophage lambda protein phosphatase by organic and oxoanion inhibitors.

Bacteriophage lambda protein phosphatase (lambdaPP) with Mn(2+) as the activating metal cofactor was studied using phosphatase inhibition kinetics and electron paramagnetic resonance (EPR) spectroscopy. Orthophosphate and the oxoanion analogues orthovanadate, tungstate, molybdate, arsenate, and sulfate were shown to inhibit the phosphomonoesterase activity of lambdaPP, albeit with inhibition constants (K(i)) that range over 5 orders of magnitude. In addition, small organic anions were tested as inhibitors. Phosphonoacetohydroxamic acid (PhAH) was found to be a strong competitive inhibitor (K(i) = 5.1 +/- 1.6 microM) whereas phosphonoacetic acid (K(i) = 380 +/- 45 microM) and acetohydroxamic acid (K(i) > 75 mM) modestly inhibited lambdaPP. Low-temperature EPR spectra of Mn(2+)-reconstituted lambdaPP in the presence of oxoanions and PhAH demonstrate that inhibitor binding decreases the spin-coupling constant, J, compared to the native enzyme. This suggests a change in the bridging interaction between Mn(2+) ions of the dimer due to protonation or replacement of a bridging ligand. Inhibitor binding also induces several spectral shifts. Hyperfine splitting characteristic of a spin-coupled (Mn(2+))(2) dimer is most prominent upon the addition of orthovanadate (K(i) = 0.70 +/- 0.20 microM) and PhAH, indicating that these inhibitors tightly interact with the (Mn(2+))(2) form of lambdaPP. These EPR and inhibition kinetic results are discussed in the context of establishing a common mechanism for the hydrolysis of phosphate esters by lambdaPP and other serine/threonine protein phosphatases.