KatG from Salmonella Typhimurium is a peroxynitritase

Pathogenic bacteria elicit protective responses to oxidative and nitrosative stresses. Although such responses are generally distinct, it was recently reported in Mycobacterium tuberculosis that catalase-peroxidase (KatG), a classical defence against peroxides, also exhibits peroxynitritase activity. Here, the katG gene from Salmonella Typhimurium was cloned and protein purified and characterised. An increase in the rate of decomposition of peroxynitrite was observed for KatG from the enterobacterium with a second-order rate constant of 4.2 × 10⁴ M⁻¹ s⁻¹ at pH 7.4, 25 °C. This enzyme was able to reduce dihydrorhodamine oxidation by peroxynitrite to ∼83%. Given the peroxynitritase activity demonstrated here it is likely that KatG may play a wider role in the detoxification of oxidative stresses than previously thought.     

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.     

Multiple thioredoxin-mediated routes to detoxify hydroperoxides in Mycobacterium tuberculosis

Drug resistance and virulence of Mycobacterium tuberculosis are in part related to the pathogen's antioxidant defense systems. KatG(-) strains are resistant to the first line tuberculostatic isoniazid but need to compensate their catalase deficiency by alternative peroxidase systems to stay virulent. So far, only NADH-driven and AhpD-mediated hydroperoxide reduction by AhpC has been implicated as such virulence-determining mechanism. We here report on two novel pathways which underscore the importance of the thioredoxin system for antioxidant defense in M. tuberculosis: (i) NADPH-driven hydroperoxide reduction by AhpC that is mediated by thioredoxin reductase and thioredoxin C and (ii) hydroperoxide reduction by the atypical peroxiredoxin TPx that equally depends on thioredoxin reductase but can use both, thioredoxin B and C. Kinetic analyses with different hydroperoxides including peroxynitrite qualify the redox cascade comprising thioredoxin reductase, thioredoxin C, and TPx as the most efficient system to protect M. tuberculosis against oxidative and nitrosative stress in situ.     

Spin trapping investigation of peroxide- and isoniazid-induced radicals in Mycobacterium tuberculosis catalase-peroxidase

A new approach, the immuno-spin trapping assay, used a novel rabbit polyclonal anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antiserum to detect protein radical-derived DMPO nitrone adducts in the hemoprotein Mycobacterium tuberculosis catalase-peroxidase (KatG). This work demonstrates that the formation of protein nitrone adducts is dependent on the concentrations of tert-BuOOH and DMPO as shown by Western blotting and an enzyme-linked immunosorbent assay (ELISA). We have also detected protein-protein cross-links formed during turnover of Mtb KatG driven by tert-butyl peroxide ( tert-BuOOH) or enzymatic generation of hydrogen peroxide. DMPO inhibits this dimerization due to its ability to trap the amino acid radicals responsible for the cross-linkage. Chemical modifications by tyrosine and tryptophan blockage suggest that tyrosine residues are one site of formation of the dimers. The presence of the tuberculosis drug isoniazid (INH) also prevented cross-linking as a result of its competition for the protein radical. Protein-DMPO nitrone adducts were also generated by a continuous flux of hydrogen peroxide. These findings demonstrated that the protein-based radicals were formed not only during Mtb KatG turnover with alkyl peroxides but also in the presence of hydrogen peroxide. Furthermore, the formation of protein-DMPO nitrone adducts was accelerated in the presence of isoniazid.     

Resonance Raman spectroscopy of Compound II and its decay in Mycobacterium tuberculosis catalase-peroxidase KatG and its isoniazid resistant mutant S315T

The reaction of Mycobacterium tuberculosis KatG and the mutant KatG(S315T) with two different organic peroxides is studied using resonance Raman spectroscopy. For the first time, an intermediate is observed in a catalase-peroxidase with vibrations that are characteristic of Compound II. The observation of this intermediate is consistent with photoreduction of Compound I and is in agreement with the formation of Compound I during the catalytic cycle of KatG. The same intermediate is detected in KatG(S315T), a mutant associated with resistance to isoniazid (INH), but with a lower yield, indicating that the organic peroxides cannot react with the heme iron in KatG(S315T) as efficiently as in wild-type KatG. Our results are consistent with catalytic competence of the S315T mutant and support the model that the S315T mutation confers antibiotic resistance by modifying the interaction between the enzyme and the drug.     

Reduced affinity for Isoniazid in the S315T mutant of Mycobacterium tuberculosis KatG is a key factor in antibiotic resistance

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 six-coordinate 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 wild-type 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.     

KatG Ser3 15Thr mutation as the main reason of isoniazid resistance in Mycobacterium tuberculosis isolated in the Novosibirsk and Kemerovo Regions

KatG Ser3 15Thr mutation is one of the main reasons of resistance to isoniazid in Mycobacterium tuberculosis isolates. The frequency rate of the above mutation among isoniazid-resistant isolates made 94% in Novosibirsk Region and 93% in Kemerovo Region. The use of PCR-restriction fragment length polymorphism (PCR-RFLP) can be regarded as an adequate method for rapid screening of isoniazid resistance in Mycobacterium tuberculosis isolated in the West-Siberian Region.     

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.     

Spectroscopic comparison of the heme active sites in WT KatG and its S315T mutant

KatG, the catalase-peroxidase from Mycobacterium tuberculosis, has been characterized by resonance Raman, electron spin resonance, and visible spectroscopies. The mutant KatG(S315T), which is found in about 50% of isoniazid-resistant clinical isolates, is also spectroscopically characterized. The electron spin resonance spectrum of ferrous nitrosyl KatG is consistent with a proximal histidine ligand. The Fe-His stretching vibration observed at 244 cm(-1) for ferrous wild-type KatG and KatG(S315T) confirms the imidazolate character of the proximal histidine in their five-coordinate high-spin complexes. The ferrous forms of wild-type KatG and KatG(S315T) are mixtures of six-coordinate low-spin and five-coordinate high-spin hemes. The optical and resonance Raman signatures of ferric wild-type KatG indicate that a majority of the heme exists in a five-coordinate high-spin state, but six-coordinate hemes are also present. At room temperature, more six-coordinate low-spin heme is observed in ferrous and ferric KatG(S315T) than in the WT enzyme. While the nature of the sixth ligand of LS ferric wild-type KatG is not completely clear, visible, resonance Raman, and electron spin resonance data of KatG(S315T) indicate that its sixth ligand is a neutral nitrogen donor. Possible effects of these differences on enzyme activity are discussed.     

Hydrogen peroxide-mediated isoniazid activation catalyzed by Mycobacterium tuberculosis catalase-peroxidase (KatG) and its S315T mutant

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.     

Modification of the active site of Mycobacterium tuberculosis KatG after disruption of the Met-Tyr-Trp cross-linked adduct

Mycobacterium tuberculosis catalase-peroxidase (Mtb KatG) is a bifunctional enzyme that possesses both catalase and peroxidase activities and is responsible for the activation of the antituberculosis drug isoniazid. Mtb KatG contains an unusual adduct in its distal heme-pocket that consists of the covalently linked Trp107, Tyr229, and Met255. The KatG(Y229F) mutant lacks this adduct and has decreased steady-state catalase activity and enhanced peroxidase activity. In order to test a potential structural role of the adduct that supports catalase activity, we have used resonance Raman spectroscopy to probe the local heme environment of KatG(Y229F). In comparison to wild-type KatG, resting KatG(Y229F) contains a significant amount of 6-coordinate, low-spin heme and a more planar heme. Resonance Raman spectroscopy of the ferrous-CO complex of KatG(Y229F) suggest a non-linear Fe-CO binding geometry that is less tilted than in wild-type KatG. These data provide evidence that the Met-Tyr-Trp adduct imparts structural stability to the active site of KatG that seems to be important for sustaining catalase activity.     

Evidence for radical formation at Tyr-353 in Mycobacterium tuberculosis catalase-peroxidase (KatG)

Mycobacterium tuberculosis KatG is a heme-containing catalase-peroxidase responsible for activation, through its peroxidase cycle, of the front line antituberculosis antibiotic isoniazid (isonicotinic acid hydrazide). Formation of Compound I (oxyferryl heme-porphyrin pi-cation radical), the classical peroxidase intermediate generated when the resting enzyme turns over with alkyl peroxides, is rapidly followed by production of a protein-centered tyrosyl radical in this enzyme. In our efforts to identify the residue at which this radical is formed, nitric oxide was used as a radical scavenging reagent. Quenching of the tyrosyl radical generated in the presence of NO was shown using electron paramagnetic resonance spectroscopy, and formation of nitrotyrosine was confirmed by proteolytic digestion followed by high performance liquid chromatography analysis of the NO-treated enzyme. These results are consistent with formation of nitrosyltyrosine by addition of NO to tyrosyl radical and oxidation of this intermediate to nitrotyrosine. Two predominant nitrotyrosine-containing peptides were identified that were purified and sequenced by Edman degradation. Both peptides were derived from the same M. tuberculosis KatG sequence spanning residues 346-356 with the amino acid sequence SPAGAWQYTAK, and both peptides contained nitrotyrosine at residue 353. Some modification of Trp-351 most probably into nitrosotryptophan was also found in one of the two peptides. Control experiments using denatured KatG or carried out in the absence of peroxide did not produce nitrotyrosine. In the mutant enzyme KatG(Y353F), which was constructed using site-directed mutagenesis, a tyrosyl radical was also formed upon turnover with peroxide but in poor yield compared with wild-type KatG. Residue Tyr-353 is unique to M. tuberculosis KatG and may play a special role in the function of this enzyme.     

Role of the Met-Tyr-Trp cross-link in Mycobacterium tuberculosis catalase-peroxidase (KatG) as revealed by KatG(M255I)

Catalase-peroxidases (KatGs) are bifunctional enzymes possessing both catalase and peroxidase activities. Four crystal structures of different KatGs revealed the presence of a novel Met-Tyr-Trp cross-link which has been suggested to impart catalatic activity to the KatGs. To decipher the individual roles of the two cross-links in the Met-Tyr-Trp adduct, we have focused on recombinant Mycobacterium tuberculosis KatG(M255I). UV-visible spectroscopic and mass spectrometric studies of the peptide fragments resulting from tryptic digestion of KatG(M255I) confirmed the presence of the single Tyr-Trp cross-link, as well as a 2e- oxidized form which is postulated to be an intermediate generated during Met-Tyr-Trp cross-link formation. KatG(M255I) lacking the Tyr-Trp cross-link was also prepared, and incubation with peroxyacetic acid, but not 2-methyl-1-phenyl-2-propyl hydroperoxide, resulted in complete formation of the Tyr-Trp cross-link. A mechanism for Tyr-Trp autocatalytic formation by KatG compound I is proposed from these studies. Optical stopped-flow studies with KatG(M255I) were performed, allowing characterization of compounds I, II, and III. Interestingly, two compound II intermediates were identified: (KatG*)(Por)Fe(III)-OH, where KatG* represents a protein-based radical, and oxoferryl (KatG)(Por)Fe(IV)=O. Insight into the contributions of the individual Tyr-Trp and Met-Tyr cross-links to catalase activity is presented, as is the overall contribution of the Met-Tyr-Trp cross-link to the structure-function-spectroscopy relationship and catalase-peroxidase mechanism in KatG.     

The Met-Tyr-Trp cross-link in Mycobacterium tuberculosis catalase-peroxidase (KatG): autocatalytic formation and effect on enzyme catalysis and spectroscopic properties

Catalase-peroxidases (KatG) are bifunctional enzymes possessing both catalase and peroxidase activities. Three crystal structures of different KatGs revealed the presence of a novel Met-Tyr-Trp cross-link that has been suggested to impart catalatic activity to the KatGs. High-performance liquid chromatographic separation of the peptide fragments resulting from tryptic digestion of recombinant Mycobacterium tuberculosis WT KatG identified a peptide with unusual UV-visible spectroscopic features attributable to the Met(255)-Tyr(229)-Trp(107) cross-link, whose structure was confirmed by mass spectrometry. WT KatG lacking the Met-Tyr-Trp cross-link was prepared, making possible studies of its formation under oxidizing conditions that generate either compound I (peroxyacetic acid, PAA) or compound II (2-methyl-1-phenyl-2-propyl hydroperoxide, MPPH). Incubation of this "cross-link-free" WT KatG with PAA revealed complete formation of the Met-Tyr-Trp structure after six equivalents of peracid were added, whereas MPPH was unable to promote cross-link formation. A mechanism for Met-Tyr-Trp autocatalytic formation by KatG compound I is proposed from these studies. Optical stopped-flow studies of WT KatG and KatG(Y229F), a mutant in which the cross-link cannot be formed, were performed with MPPH and revealed an unusual compound II spectrum for WT KatG, best described as (P.)Fe(III), where P. represents a protein-based radical. This contrasts with the oxoferryl compound II spectrum observed for KatG(Y229F) under identical conditions. The structure-function-spectroscopy relationship in KatG is discussed with relevance to the role that the Met-Tyr-Trp cross-link plays in the catalase-peroxidase mechanism.     

Redox potential measurements of the Mycobacterium tuberculosis heme protein KatG and the isoniazid-resistant enzyme KatG(S315T): insights into isoniazid activation

Mycobacterium tuberculosis KatG is a multifunctional heme enzyme responsible for activation of the antibiotic isoniazid. A KatG(S315T) point mutation is found in >50% of isoniazid-resistant clinical isolates. Since isoniazid activation is thought to involve an oxidation reaction, the redox potential of KatG was determined using cyclic voltammetry, square wave voltammetry, and spectroelectrochemical titrations. Isoniazid activation may proceed via a cytochrome P450-like mechanism. Therefore, the possibility that substrate binding by KatG leads to an increase in the heme redox potential and the possibility that KatG(S315T) confers isoniazid resistance by altering the redox potential were examined. Effects of the heme spin state on the reduction potentials of KatG and KatG(S315T) were also determined. Assessment of the Fe(3+)/Fe(2+) couple gave a midpoint potential of ca. -50 mV for both KatG and KatG(S315T). In contrast to cytochrome P450s, addition of substrate had no significant effect on either the KatG or KatG(S315T) redox potential. Conversion of the heme to a low-spin configuration resulted in a -150 to -200 mV shift of the KatG and KatG(S315T) redox potentials. These results suggest that isoniazid resistance conferred by KatG(S315T) is not mediated through changes in the heme redox potential. The redox potentials of isoniazid were also determined using cyclic and square wave voltammetry, and the results provide evidence that the ferric KatG and KatG(S315T) midpoint potentials are too low to promote isoniazid oxidation without formation of a high-valent enzyme intermediate such as compounds I and II or oxyferrous KatG.     

Role of KatG catalase-peroxidase in mycobacterial pathogenesis: countering the phagocyte oxidative burst

Reactive nitrogen species (RNS) play an essential role in host defence against Mycobacterium tuberculosis (MTB) in the mouse model of tuberculosis (TB), as evidenced by the increased susceptibility of mice deficient in the inducible isoform of nitric oxide synthase (NOS2). In contrast, the role of reactive oxygen species (ROS) in protection against MTB is less clear, and mice defective in the ROS-generating phagocyte NADPH oxidase (Phox) are relatively resistant. This suggests that MTB might possess efficient mechanisms to evade or counter the phagocyte oxidative burst, effectively masking the impact of this host defence mechanism. In order to assess the role of ROS detoxification pathways in MTB virulence, we generated a katG null mutant of MTB, deficient in the KatG catalase-peroxidase-peroxynitritase, and evaluated the mutant's ability to replicate and persist in macrophages and mice. Although markedly attenuated in wild-type C57Bl/6 mice and NOS2(-/-) mice, the DeltakatG MTB strain was indistinguishable from wild-type MTB in its ability to replicate and persist in gp91(Phox-/-) mice lacking the gp91 subunit of NADPH oxidase. Similar observations were made with murine bone marrow macrophages infected ex vivo: growth of the DeltakatG MTB strain was impaired in macrophages from C57Bl/6 and NOS2(-/-) mice, but indistinguishable from wild-type MTB in gp91(Phox-/-) macrophages. These results indicate that the major role of KatG in MTB pathogenesis is to catabolize the peroxides generated by the phagocyte NADPH oxidase; in the absence of this host antimicrobial mechanism, KatG is apparently dispensable.     

Conformational differences in Mycobacterium tuberculosis catalase-peroxidase KatG and its S315T mutant revealed by resonance Raman spectroscopy

KatG from Mycobacterium tuberculosis is a heme-containing catalase-peroxidase, which belongs to the class I peroxidases and is important for activation of the prodrug isoniazid (INH), a front-line antituberculosis drug. In many clinical isolates, resistance to INH has been linked to mutations on the katG gene, and the most prevalent mutation, S315T, suggests that modification of the heme pocket has occurred. Electronic absorption and resonance Raman spectra of ferric wild-type (WT) KatG and its INH-resistant mutant KatG(S315T) at different pH values and their complexes with INH and benzohydroxamic acid (BHA) are reported. At neutral pH, a quantum mechanically mixed spin state (QS) is revealed, which coexists with five-coordinate and six-coordinate high-spin hemes in WT KatG. The QS heme is the major species in KatG(S315T). Addition of either INH or BHA to KatG induces only minor changes in the resonance Raman spectra, indicating that both compounds do not directly interact with the heme iron. New vibrational modes are observed at 430, 473, and 521 cm(-1), and these modes are indicative of a change in conformation in the KatG heme pocket. The intensity of these modes and the relative population of the QS heme are stable in KatG(S315T) but not in the WT enzyme. This indicates that there are differences in heme pocket stability between WT KatG and KatG(S315T). We will discuss the stabilization of the QS heme and propose a model for the inhibition of INH oxidation by KatG(S315T).     

Action mechanism of antitubercular isoniazid. Activation by Mycobacterium tuberculosis KatG, isolation, and characterization of inha inhibitor

Activation of the antitubercular isoniazid (INH) by the Mycobacterium tuberculosis KatG produces an inhibitor for enoyl reductase (InhA). The mechanism for INH activation remains poorly understood, and the inhibitor has never been isolated. We have purified the InhA-inhibitor complex generated in the M. tuberculosis KatG-catalyzed INH activation. The complex exhibited a 278-nm absorption peak and a shoulder around 326 nm with a characteristic A(326)/A(278) ratio of 0.16. The complex was devoid of enoyl reductase activity. The inhibitor noncovalently binds to InhA with a K(d) < 0.4 nM and can be dissociated from denatured InhA for chromatographic isolation. The free inhibitor showed absorption peaks at 326 (epsilon(326) 6900 M(-1) cm(-1)) and 260 nm (epsilon(260) 27,000 M(-1) cm(-1)). The inactive complex can be reconstituted from InhA and the isolated inhibitor. The InhA inhibitor from the KatG-catalyzed INH activation was identical to that from a slow, KatG-independent, Mn(2+)-mediated reaction based on high pressure liquid chromatography analysis and absorption and mass spectral characteristics. By monitoring the formation of the InhA-inhibitor complex, we have found that manganese is not essential to the INH activation by M. tuberculosis KatG. Furthermore, the formation of the InhA inhibitor in the KatG reaction was independent of InhA.